Nucleic acids and proteins from streptococcus groups A and B

ABSTRACT

The invention provides proteins from group B  streptococcus  ( Streptococcus agalactiae ) and group A  streptococcus  ( Streptococcus pyogenes ), including amino acid sequences and the corresponding nucleotide sequences. Data are given to show that the proteins are useful antigens for vaccines, immunogenic compositions, and/or diagnostics. The proteins are also targets for antibiotics.

This application is a continuation of Ser. No. 14/751,790, now issued as U.S. Pat. No. 9,840,538, which is a continuation of Ser. No. 14/615,108 filed Feb. 5, 2015. Ser. No. 14/615,108 is a continuation of Ser. No. 13/598,657 filed on Aug. 30, 2012, now abandoned. Ser. No. 13/598,657 is a division of Ser. No. 11/434,203 filed on May 16, 2006, now issued as U.S. Pat. No. 8,431,139, which is a continuation of Ser. No. 10/415,182 filed Apr. 28, 2003, now issued as U.S. Pat. No. 7,939,087. Ser. No. 10/415,182 is a National Stage application of PCT application PCT/GB01/04789, which was filed Oct. 29, 2001 and published in English under PCT Article 21(2) on May 2, 2002. PCT/GB01/04789 claims the benefit of Serial No. GB0026333.5 filed Oct. 27, 2000, Serial No. GB0028727.6 filed Nov. 24, 2000, and Serial No. GB0105640.7 filed Mar. 7, 2001. Each of these applications and all the other documents cited herein are incorporated by reference in their entireties.

This application incorporates by reference the contents of a 22.0 MB file created on Jun. 25, 2015 and submitted herewith, which is the sequence listing for this application.

TECHNICAL FIELD

This invention relates to nucleic acid and proteins from the bacteria Streptococcus agalactiae (GBS) and Streptococcus pyogenes (GAS).

BACKGROUND ART

Once thought to infect only cows, the Gram-positive bacterium Streptococcus agalactiae (or “group B streptococcus”, abbreviated to “GBS”) is now known to cause serious disease, bacteremia and meningitis, in immunocompromised individuals and in neonates. There are two types of neonatal infection. The first (early onset, usually within 5 days of birth) is manifested by bacteremia and pneumonia. It is contracted vertically as a baby passes through the birth canal. GBS colonises the vagina of about 25% of young women, and approximately 1% of infants born via a vaginal birth to colonised mothers will become infected. Mortality is between 50-70%. The second is a meningitis that occurs 10 to 60 days after birth. If pregnant women are vaccinated with type III capsule so that the infants are passively immunised, the incidence of the late onset meningitis is reduced but is not entirely eliminated.

The “B” in “GBS” refers to the Lancefield classification, which is based on the antigenicity of a carbohydrate which is soluble in dilute acid and called the C carbohydrate. Lancefield identified 13 types of C carbohydrate, designated A to O, that could be serologically differentiated. The organisms that most commonly infect humans are found in groups A, B, D, and G. Within group B, strains can be divided into 8 serotypes (Ia, Ib, Ia/c, II, III, IV, V, and VI) based on the structure of their polysaccharide capsule.

Group A streptococcus (“GAS”, S. pyogenes) is a frequent human pathogen, estimated to be present in between 5-15% of normal individuals without signs of disease. When host defences are compromised, or when the organism is able to exert its virulence, or when it is introduced to vulnerable tissues or hosts, however, an acute infection occurs. Diseases include puerperal fever, scarlet fever, erysipelas, pharyngitis, impetigo, necrotising fasciitis, myositis and streptococcal toxic shock syndrome.

S. pyogenes is typically treated using antibiotics. Although S. agalactiae is inhibited by antibiotics, however, it is not killed by penicillin as easily as GAS. Prophylactic vaccination is thus preferable.

Current GBS vaccines are based on polysaccharide antigens, although these suffer from poor immunogenicity. Anti-idiotypic approaches have also been used (e.g. WO99/54457). There remains a need, however, for effective adult vaccines against S. agalactiae infection. There also remains a need for vaccines against S. pyogenes infection.

It is an object of the invention to provide proteins which can be used in the development of such vaccines. The proteins may also be useful for diagnostic purposes, and as targets for antibiotics.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1 to 85, 119-188, 238, and 239 show SDS-PAGE analysis of total cell extracts from cultures of recombinant E. coli expressing GBS proteins of the invention. Lane 1 in each gel (except for FIG. 185) contains molecular weight markers. These are 94, 67, 43, 30, 20.1, and 14.4 kDa (except for FIGS. 7, 8, 10, 11, 13, 14, 15, and 119-170, which use 250, 150, 100, 75, 50, 37, 25, 15 & 10 kDa).

FIG. 86A shows the pDEST15 vector. FIG. 86B shows the pDEST17-1 vector.

FIGS. 87 to 118 and 247 to 319 show protein characterization data for various proteins of the invention.

FIGS. 189 to 237 and 240 to 246 show SDS-PAGE analysis of purified GBS proteins of the invention. The left-hand lane contains molecular weight markers. These are 94, 67, 43, 30, 20.1, and 14.4 kDa.

DETAILED DESCRIPTION

The invention provides proteins comprising the S. agalactiae amino acid sequences disclosed herein, and proteins comprising the S. pyogenes amino acid sequences disclosed herein. These amino acid sequences are the even SEQ ID NOS: between 1 and 10960.

The invention provides proteins comprising the S. agalactiae amino acid sequence disclosed in the example, and proteins comprising the S. pyogenes amino acid sequence disclosed in the example. These amino acid sequences are SEQ ID NOS: 4210 and 4212, respectively.

It also provides proteins comprising amino acid sequences having sequence identity to the S. agalactiae amino acid sequence disclosed in the example, and proteins comprising amino acid sequences having sequence identity to the S. pyogenes amino acid sequence disclosed in the example. Depending on the particular sequence, the degree of sequence identity is preferably greater than 50% (e.g. 60%, 70%, 80%, 90%, 95%, 99% or more). These proteins include homologs, orthologs, allelic variants and functional mutants. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence. Identity between proteins is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1.

Preferred proteins of the invention are GBS1 to GBS689 (see Table IV).

The invention further provides proteins comprising fragments of the S. agalactiae amino acid sequence disclosed in the example, and proteins comprising fragments of the S. pyogenes amino acid sequence disclosed in the example. The fragments should comprise at least n consecutive amino acids from the sequences and, depending on the particular sequence, n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more). Preferably the fragments comprise one or more epitopes from the sequence. Other preferred fragments are (a) the N-terminal signal peptides of the proteins disclosed in the example, (b) the proteins disclosed in the example, but without their N-terminal signal peptides, (c) fragments common to the related GAS and GBS proteins disclosed in the example, and (d) the proteins disclosed in the example, but without their N-terminal amino acid residue.

The proteins of the invention can, of course, be prepared by various means (e.g. recombinant expression, purification from GAS or GBS, chemical synthesis etc.) and in various forms (e.g. native, fusions, glycosylated, non-glycosylated etc.). They are preferably prepared in substantially pure form (i.e. substantially free from other streptococcal or host cell proteins) or substantially isolated form. Proteins of the invention are preferably streptococcal proteins.

According to a further aspect, the invention provides antibodies which bind to these proteins. These may be polyclonal or monoclonal and may be produced by any suitable means (e.g. by recombinant expression). To increase compatibility with the human immune system, the antibodies may be chimeric or humanised (e.g. Breedveld (2000) Lancet 355(9205):735-740; Gorman & Clark (1990) Semin. Immunol. 2:457-466), or fully human antibodies may be used. The antibodies may include a detectable label (e.g. for diagnostic assays).

According to a further aspect, the invention provides nucleic acid comprising the S. agalactiae nucleotide sequences disclosed herein, and nucleic acid comprising the S. pyogenes nucleotide sequences disclosed herein. These nucleic acid sequences are the odd SEQ ID NOS: between 1 and 10966.

According to a further aspect, the invention provides nucleic acid comprising the S. agalactiae nucleotide sequence disclosed in the example, and nucleic acid comprising the S. pyogenes nucleotide sequence disclosed in the example. These nucleic acid sequences are SEQ ID NOS: 4209 and 4211, respectively.

In addition, the invention provides nucleic acid comprising nucleotide sequences having sequence identity to the S. agalactiae nucleotide sequence disclosed in the example, and nucleic acid comprising nucleotide sequences having sequence identity to the S. pyogenes nucleotide sequence disclosed in the example. Identity between sequences is preferably determined by the Smith-Waterman homology search algorithm as described above.

Furthermore, the invention provides nucleic acid which can hybridise to the S. agalactiae nucleic acid disclosed in the example, and nucleic acid which can hybridise to the S. pyogenes nucleic acid disclosed in the example preferably under ‘high stringency’ conditions (e.g. 65° C. in 0.1×SSC, 0.5% SDS solution).

Nucleic acid comprising fragments of these sequences are also provided. These should comprise at least n consecutive nucleotides from the S. agalactiae or S. pyogenes sequences and, depending on the particular sequence, n is 10 or more (e.g. 12, 14, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more). The fragments may comprise sequences which are common to the related GAS and GBS sequences disclosed in the examples.

According to a further aspect, the invention provides nucleic acid encoding the proteins and protein fragments of the invention.

The invention also provides: nucleic acid comprising nucleotide sequence SEQ ID NO:10967; nucleic acid comprising nucleotide sequences having sequence identity to SEQ ID NO: 10967; nucleic acid which can hybridise to SEQ ID NO: 10967 (preferably under ‘high stringency’ conditions); nucleic acid comprising a fragment of at least n consecutive nucleotides from SEQ ID NO: 10967, wherein n is 10 or more e.g. 12, 14, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 10000, 100000, 1000000 or more.

The invention also provides: nucleic acid comprising nucleotide sequence SEQ ID NO:10967, nucleic acid comprising nucleotide sequences having sequence identity to SEQ ID NO:10967; nucleic

Nucleic acids of the invention can be used in hybridisation reactions (e.g. Northern or Southern blots, or in nucleic acid microarrays or ‘gene chips’) and amplification reactions (e.g. PCR, SDA, SSSR, LCR, TMA, NASBA etc.) and other nucleic acid techniques.

It should also be appreciated that the invention provides nucleic acid comprising sequences complementary to those described above (e.g. for antisense or probing, or for use as primers).

Nucleic acid according to the invention can, of course, be prepared in many ways (e.g. by chemical synthesis, from genomic or cDNA libraries, from the organism itself etc.) and can take various forms (e.g. single stranded, double stranded, vectors, primers, probes, labelled etc.). The nucleic acid is preferably in substantially isolated form.

Nucleic acid according to the invention may be labelled e.g. with a radioactive or fluorescent label. This is particularly useful where the nucleic acid is to be used in nucleic acid detection techniques e.g. where the nucleic acid is a primer or as a probe for use in techniques such as PCR, LCR, TMA, NASBA etc.

In addition, the term “nucleic acid” includes DNA and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA) etc.

According to a further aspect, the invention provides vectors comprising nucleotide sequences of the invention (e.g. cloning or expression vectors) and host cells transformed with such vectors.

According to a further aspect, the invention provides compositions comprising protein, antibody, and/or nucleic acid according to the invention. These compositions may be suitable as immunogenic compositions, for instance, or as diagnostic reagents, or as vaccines.

The invention also provides nucleic acid, protein, or antibody according to the invention for use as medicaments (e.g. as immunogenic compositions or as vaccines) or as diagnostic reagents. It also provides the use of nucleic acid, protein, or antibody according to the invention in the manufacture of: (i) a medicament for treating or preventing disease and/or infection caused by streptococcus; (ii) a diagnostic reagent for detecting the presence of streptococcus or of antibodies raised against streptococcus; and/or (iii) a reagent which can raise antibodies against streptococcus. Said streptococcus may be any species, group or strain, but is preferably S. agalactiae, especially serotype III or V, or S. pyogenes. Said disease may be bacteremia, meningitis, puerperal fever, scarlet fever, erysipelas, pharyngitis, impetigo, necrotising fasciitis, myositis or toxic shock syndrome.

The invention also provides a method of treating a patient, comprising administering to the patient a therapeutically effective amount of nucleic acid, protein, and/or antibody of the invention. The patient may either be at risk from the disease themselves or may be a pregnant woman (‘maternal immunisation’ e.g. Glezen & Alpers (1999) Clin. Infect. Dis. 28:219-224).

Administration of protein antigens is a preferred method of treatment for inducing immunity.

Administration of antibodies of the invention is another preferred method of treatment. This method of passive immunisation is particularly useful for newborn children or for pregnant women. This method will typically use monoclonal antibodies, which will be humanised or fully human.

The invention also provides a kit comprising primers (e.g. PCR primers) for amplifying a template sequence contained within a Streptococcus (e.g. S. pyogenes or S. agalactiae) nucleic acid sequence, the kit comprising a first primer and a second primer, wherein the first primer is substantially complementary to said template sequence and the second primer is substantially complementary to a complement of said template sequence, wherein the parts of said primers which have substantial complementarity define the termini of the template sequence to be amplified. The first primer and/or the second primer may include a detectable label (e.g. a fluorescent label).

The invention also provides a kit comprising first and second single-stranded oligonucleotides which allow amplification of a Streptococcus template nucleic acid sequence contained in a single- or double-stranded nucleic acid (or mixture thereof), wherein: (a) the first oligonucleotide comprises a primer sequence which is substantially complementary to said template nucleic acid sequence; (b) the second oligonucleotide comprises a primer sequence which is substantially complementary to the complement of said template nucleic acid sequence; (c) the first oligonucleotide and/or the second oligonucleotide comprise(s) sequence which is not complementary to said template nucleic acid; and (d) said primer sequences define the termini of the template sequence to be amplified. The non-complementary sequence(s) of feature (c) are preferably upstream of (i.e. 5′ to) the primer sequences. One or both of these (c) sequences may comprise a restriction site (e.g. EP-B-0509612) or a promoter sequence (e.g. EP-B-0505012). The first oligonucleotide and/or the second oligonucleotide may include a detectable label (e.g. a fluorescent label).

The template sequence may be any part of a genome sequence (e.g. SEQ ID NO:10967). For example, it could be a rRNA gene (e.g. Turenne et al. (2000) J. Clin. Microbiol. 38:513-520; SEQ ID NOS: 12018-12024 herein) or a protein-coding gene. The template sequence is preferably specific to GBS.

The invention also provides a computer-readable medium (e.g. a floppy disk, a hard disk, a CD-ROM, a DVD etc.) and/or a computer database containing one or more of the sequences in the sequence listing. The medium preferably contains one or both of SEQ ID NO:10967.

The invention also provides a hybrid protein represented by the formula NH₂-A-[-X-L-]_(n)-B—COOH, wherein X is a protein of the invention, L is an optional linker amino acid sequence, A is an optional N-terminal amino acid sequence, B is an optional C-terminal amino acid sequence, and n is an integer greater than 1. The value of n is between 2 and x, and the value of x is typically 3, 4, 5, 6, 7, 8, 9 or 10. Preferably n is 2, 3 or 4; it is more preferably 2 or 3; most preferably, n=2. For each n instances, —X— may be the same or different. For each n instances of [-X-L-], linker amino acid sequence -L- may be present or absent. For instance, when n=2 the hybrid may be NH₂—X₁-L₁-X₂-L₂-COOH, NH₂—X₁-X₂—COOH, NH₂—X₁-L₁-X₂—COOH, NH₂—X₁-X₂-L₂-COOH, etc Linker amino acid sequence(s) -L- will typically be short (e.g. 20 or fewer amino acids i.e. 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include short peptide sequences which facilitate cloning, poly-glycine linkers (i.e. Gly_(n) where n=2, 3, 4, 5, 6, 7, 8, 9, 10 or more), and histidine tags (i.e. His_(n) where n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable linker amino acid sequences will be apparent to those skilled in the art. -A- and —B— are optional sequences which will typically be short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include leader sequences to direct protein trafficking, or short peptide sequences which facilitate cloning or purification (e.g. histidine tags i.e. His_(n) where n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable N-terminal and C-terminal amino acid sequences will be apparent to those skilled in the art. In some embodiments, each X will be a GBS sequence; in others, mixtures of GAS and GBS will be used.

According to further aspects, the invention provides various processes.

A process for producing proteins of the invention is provided, comprising the step of culturing a host cell of to the invention under conditions which induce protein expression.

A process for producing protein or nucleic acid of the invention is provided, wherein the protein or nucleic acid is synthesised in part or in whole using chemical means.

A process for detecting polynucleotides of the invention is provided, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridising conditions to form duplexes; and (b) detecting said duplexes.

A process for detecting Streptococcus in a biological sample (e.g. blood) is also provided, comprising the step of contacting nucleic acid according to the invention with the biological sample under hybridising conditions. The process may involve nucleic acid amplification (e.g. PCR, SDA, SSSR, LCR, TMA, NASBA etc.) or hybridisation (e.g. microarrays, blots, hybridisation with a probe in solution etc.). PCR detection of Streptococcus in clinical samples, in particular S. pyogenes, has been reported [see e.g. Louie et al. (2000) CMAJ 163:301-309; Louie et al. (1998) J. Clin. Microbiol. 36:1769-1771]. Clinical assays based on nucleic acid are described in general in Tang et al. (1997) Clin. Chem. 43:2021-2038.

A process for detecting proteins of the invention is provided, comprising the steps of: (a) contacting an antibody of the invention with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes.

A process for identifying an amino acid sequence is provided, comprising the step of searching for putative open reading frames or protein-coding regions within a genome sequence of S. agalactiae. This will typically involve in silico searching the sequence for an initiation codon and for an in-frame termination codon in the downstream sequence. The region between these initiation and termination codons is a putative protein-coding sequence. Typically, all six possible reading frames will be searched. Suitable software for such analysis includes ORFFINDER (NCBI), GENEMARK [Borodovsky & McIninch (1993) Computers Chem. 17:122-133), GLIMMER [Salzberg et al. (1998) Nucleic Acids Res. 26:544-548; Salzberg et al. (1999) Genomics 59:24-31; Delcher et al. (1999) Nucleic Acids Res. 27:4636-4641], or other software which uses Markov models [e.g. Shmatkov et al. (1999) Bioinformatics 15:874-876]. The invention also provides a protein comprising the identified amino acid sequence. These proteins can then be expressed using conventional techniques.

The invention also provides a process for determining whether a test compound binds to a protein of the invention. If a test compound binds to a protein of the invention and this binding inhibits the life cycle of the GBS bacterium, then the test compound can be used as an antibiotic or as a lead compound for the design of antibiotics. The process will typically comprise the steps of contacting a test compound with a protein of the invention, and determining whether the test compound binds to said protein. Preferred proteins of the invention for use in these processes are enzymes (e.g. tRNA synthetases), membrane transporters and ribosomal proteins. Suitable test compounds include proteins, polypeptides, carbohydrates, lipids, nucleic acids (e.g. DNA, RNA, and modified forms thereof), as well as small organic compounds (e.g. MW between 200 and 2000 Da). The test compounds may be provided individually, but will typically be part of a library (e.g. a combinatorial library). Methods for detecting a binding interaction include NMR, filter-binding assays, gel-retardation assays, displacement assays, surface plasmon resonance, reverse two-hybrid etc. A compound which binds to a protein of the invention can be tested for antibiotic activity by contacting the compound with GBS bacteria and then monitoring for inhibition of growth. The invention also provides a compound identified using these methods.

The invention also provides a composition comprising a protein or the invention and one or more of the following antigens:

-   -   a protein antigen from Helicobacter pylori such as VacA, CagA,         NAP, HopX, HopY [e.g. WO98/04702] and/or urease.     -   a protein antigen from N. meningitidis serogroup B, such as         those in WO99/24578, WO99/36544, WO99/57280, WO00/22430,         Tettelin et al. (2000) Science 287:1809-1815, Pizza et         al. (2000) Science 287:1816-1820 and WO96/29412, with protein         ‘287’ and derivatives being particularly preferred.     -   an outer-membrane vesicle (OMV) preparation from N. meningitidis         serogroup B, such as those disclosed in WO01/52885; Bjune et         al. (1991) Lancet 338(8775):1093-1096; Fukasawa et al. (1999)         Vaccine 17:2951-2958; Rosenqvist et al. (1998) Dev. Biol. Stand.         92:323-333 etc.     -   a saccharide antigen from N. meningitidis serogroup A, C, W135         and/or Y, such as the oligosaccharide disclosed in Costantino et         al. (1992) Vaccine 10:691-698 from serogroup C [see also         Costantino et al. (1999) Vaccine 17:1251-1263].     -   a saccharide antigen from Streptococcus pneumoniae [e.g.         Watson (2000) Pediatr Infect Dis J 19:331-332; Rubin (2000)         Pediatr Clin North Am 47:269-285, v; Jedrzejas (2001) Microbiol         Mol Biol Rev 65:187-207].     -   an antigen from hepatitis A virus, such as inactivated virus         [e.g. Bell (2000) Pediatr Infect Dis J 19:1187-1188;         Iwarson (1995) APMIS 103:321-326].     -   an antigen from hepatitis B virus, such as the surface and/or         core antigens [e.g. Gerlich et al. (1990) Vaccine 8 Suppl:S63-68         & 79-80].     -   an antigen from hepatitis C virus [e.g. Hsu et al. (1999) Clin         Liver Dis 3:901-915].     -   an antigen from Bordetella pertussis, such as pertussis         holotoxin (PT) and filamentous haemagglutinin (FHA) from B.         pertussis, optionally also in combination with pertactin and/or         agglutinogens 2 and 3 [e.g. Gustafsson et al. (1996) N. Engl. J.         Med. 334:349-355; Rappuoli et al. (1991) TIBTECH 9:232-238].     -   a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter         3 of Vaccines (1988) eds. Plotkin & Mortimer. ISBN         0-7216-1946-0] e.g. the CRM₁₉₇ mutant [e.g. Del Guidice et         al. (1998) Molecular Aspects of Medicine 19:1-70].     -   a tetanus antigen, such as a tetanus toxoid [e.g. chapter 4 of         Plotkin & Mortimer].     -   a saccharide antigen from Haemophilus influenzae B.     -   an antigen from N. gonorrhoeae [e.g. WO99/24578, WO99/36544,         WO99/57280].     -   an antigen from Chlamydia pneumoniae [e.g. PCT/IB01/01445;         Kalman et al. (1999) Nature Genetics 21:385-389; Read et         al. (2000) Nucleic Acids Res 28:1397-406; Shirai et         al. (2000) J. Infect. Dis. 181(Suppl 3):S524-S527; WO99/27105;         WO00/27994; WO00/37494].     -   an antigen from Chlamydia trachomatis [e.g. WO99/28475].     -   an antigen from Porphyromonas gingivalis [e.g. Ross et         al. (2001) Vaccine 19:4135-4142].     -   polio antigen(s) [e.g. Sutter et al. (2000) Pediatr Clin North         Am 47:287-308; Zimmerman & Spann (1999) Am Fam Physician         59:113-118, 125-126] such as IPV or OPV.     -   rabies antigen(s) [e.g. Dreesen (1997) Vaccine 15 Suppl:S2-6]         such as lyophilised inactivated virus [e.g. MMWR Morb Mortal         Wkly Rep 1998 Jan. 16; 47(1):12, 19; RabAvert™].     -   measles, mumps and/or rubella antigens [e.g. chapters 9, 10 & 11         of Plotkin & Mortimer].     -   influenza antigen(s) [e.g. chapter 19 of Plotkin & Mortimer],         such as the haemagglutinin and/or neuraminidase surface         proteins.     -   an antigen from Moraxella catarrhalis [e.g. McMichael (2000)         Vaccine 19 Suppl 1:S101-107].     -   an antigen from Staphylococcus aureus [e.g. Kuroda et al. (2001)         Lancet 357(9264):1225-1240; see also pages 1218-1219].

Where a saccharide or carbohydrate antigen is included, it is preferably conjugated to a carrier protein in order to enhance immunogenicity [e.g. Ramsay et al. (2001) Lancet 357(9251):195-196; Lindberg (1999) Vaccine 17 Suppl 2:S28-36; Conjugate Vaccines (eds. Cruse et al.) ISBN 3805549326, particularly vol. 10:48-114 etc.]. Preferred carrier proteins are bacterial toxins or toxoids, such as diphtheria or tetanus toxoids. The CRM₁₉₇ diphtheria toxoid is particularly preferred. Other suitable carrier proteins include the N. meningitidis outer membrane protein [e.g. EP-0372501], synthetic peptides [e.g. EP-0378881, EP-0427347], heat shock proteins [e.g. WO93/17712], pertussis proteins [e.g. WO98/58668; EP-0471177], protein D from H. influenzae [e.g. WO00/56360], toxin A or B from C. difficile [e.g. WO00/61761], etc. Any suitable conjugation reaction can be used, with any suitable linker where necessary.

Toxic protein antigens may be detoxified where necessary (e.g. detoxification of pertussis toxin by chemical and/or genetic means).

Where a diphtheria antigen is included in the composition it is preferred also to include tetanus antigen and pertussis antigens. Similarly, where a tetanus antigen is included it is preferred also to include diphtheria and pertussis antigens. Similarly, where a pertussis antigen is included it is preferred also to include diphtheria and tetanus antigens.

Antigens are preferably adsorbed to an aluminium salt.

Antigens in the composition will typically be present at a concentration of at least 1 μg/ml each. In general, the concentration of any given antigen will be sufficient to elicit an immune response against that antigen.

The invention also provides compositions comprising two or more proteins of the present invention. The two or more proteins may comprise GBS sequences or may comprise GAS and GBS sequences.

A summary of standard techniques and procedures which may be employed to perform the invention (e.g. to utilise the disclosed sequences for vaccination or diagnostic purposes) follows. This summary is not a limitation on the invention but, rather, gives examples that may be used, but are not required.

General

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature eg. Sambrook Molecular Cloning; A Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes I and II (D. N Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed, 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription and Translation (B. D. Hames & S. J. Higgins eds. 1984); Animal Cell Culture (R. I. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 & 155; Gene Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos eds. 1987, Cold Spring Harbor Laboratory); Mayer and Walker, eds. (1987), Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, (1987) Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N.Y.), and Handbook of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell eds 1986).

Standard abbreviations for nucleotides and amino acids are used in this specification.

Definitions

A composition containing X is “substantially free of” Y when at least 85% by weight of the total X+Y in the composition is X. Preferably, X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95% or even 99% by weight.

The term “comprising” means “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X+Y.

The term “heterologous” refers to two biological components that are not found together in nature. The components may be host cells, genes, or regulatory regions, such as promoters. Although the heterologous components are not found together in nature, they can function together, as when a promoter heterologous to a gene is operably linked to the gene. Another example is where a streptococcus sequence is heterologous to a mouse host cell. A further examples would be two epitopes from the same or different proteins which have been assembled in a single protein in an arrangement not found in nature.

An “origin of replication” is a polynucleotide sequence that initiates and regulates replication of polynucleotides, such as an expression vector. The origin of replication behaves as an autonomous unit of polynucleotide replication within a cell, capable of replication under its own control. An origin of replication may be needed for a vector to replicate in a particular host cell. With certain origins of replication, an expression vector can be reproduced at a high copy number in the presence of the appropriate proteins within the cell. Examples of origins are the autonomously replicating sequences, which are effective in yeast; and the viral T-antigen, effective in COS-7 cells.

A “mutant” sequence is defined as DNA, RNA or amino acid sequence differing from but having sequence identity with the native or disclosed sequence. Depending on the particular sequence, the degree of sequence identity between the native or disclosed sequence and the mutant sequence is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more, calculated using the Smith-Waterman algorithm as described above). As used herein, an “allelic variant” of a nucleic acid molecule, or region, for which nucleic acid sequence is provided herein is a nucleic acid molecule, or region, that occurs essentially at the same locus in the genome of another or second isolate, and that, due to natural variation caused by, for example, mutation or recombination, has a similar but not identical nucleic acid sequence. A coding region allelic variant typically encodes a protein having similar activity to that of the protein encoded by the gene to which it is being compared. An allelic variant can also comprise an alteration in the 5′ or 3′ untranslated regions of the gene, such as in regulatory control regions (eg. see U.S. Pat. No. 5,753,235).

Expression Systems

The streptococcus nucleotide sequences can be expressed in a variety of different expression systems; for example those used with mammalian cells, baculoviruses, plants, bacteria, and yeast.

i. Mammalian Systems

Mammalian expression systems are known in the art. A mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to the 5′ end of the coding sequence, and a TATA box, usually located 25-30 base pairs (bp) upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A mammalian promoter will also contain an upstream promoter element, usually located within 100 to 200 bp upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation [Sambrook et al. (1989) “Expression of Cloned Genes in Mammalian Cells.” In Molecular Cloning: A Laboratory Manual, 2nd ed.].

Mammalian viral genes are often highly expressed and have a broad host range; therefore sequences encoding mammalian viral genes provide particularly useful promoter sequences. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), and herpes simplex virus promoter. In addition, sequences derived from non-viral genes, such as the murine metallotheionin gene, also provide useful promoter sequences. Expression may be either constitutive or regulated (inducible), depending on the promoter can be induced with glucocorticoid in hormone-responsive cells.

The presence of an enhancer element (enhancer), combined with the promoter elements described above, will usually increase expression levels. An enhancer is a regulatory DNA sequence that can stimulate transcription up to 1000-fold when linked to homologous or heterologous promoters, with synthesis beginning at the normal RNA start site. Enhancers are also active when they are placed upstream or downstream from the transcription initiation site, in either normal or flipped orientation, or at a distance of more than 1000 nucleotides from the promoter [Maniatis et al. (1987) Science 236:1237; Alberts et al. (1989) Molecular Biology of the Cell, 2nd ed.]. Enhancer elements derived from viruses may be particularly useful, because they usually have a broader host range. Examples include the SV40 early gene enhancer [Dijkema et al (1985) EMBO J. 4:761] and the enhancer/promoters derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al. (1982b) Proc. Natl. Acad. Sci. 79:6777] and from human cytomegalovirus [Boshart et al. (1985) Cell 41:521]. Additionally, some enhancers are regulatable and become active only in the presence of an inducer, such as a hormone or metal ion [Sassone-Corsi and Borelli (1986) Trends Genet. 2:215; Maniatis et al. (1987) Science 236:1237].

A DNA molecule may be expressed intracellularly in mammalian cells. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.

Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in mammalian cells. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The adenovirus tripartite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells.

Usually, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3′ to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3′ terminus of the mature mRNA is formed by site-specific post-transcriptional cleavage and polyadenylation [Birnstiel et al. (1985) Cell 41:349; Proudfoot and Whitelaw (1988) “Termination and 3′ end processing of eukaryotic RNA. In Transcription and splicing (ed. B. D. Hames and D. M. Glover); Proudfoot (1989) Trends Biochem. Sci. 14:105]. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminater/polyadenylation signals include those derived from SV40 [Sambrook et al (1989) “Expression of cloned genes in cultured mammalian cells.” In Molecular Cloning: A Laboratory Manual].

Usually, the above described components, comprising a promoter, polyadenylation signal, and transcription termination sequence are put together into expression constructs. Enhancers, introns with functional splice donor and acceptor sites, and leader sequences may also be included in an expression construct, if desired. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as mammalian cells or bacteria. Mammalian replication systems include those derived from animal viruses, which require trans-acting factors to replicate. For example, plasmids containing the replication systems of papovaviruses, such as SV40 [Gluzman (1981) Cell 23:175] or polyomavirus, replicate to extremely high copy number in the presence of the appropriate viral T antigen. Additional examples of mammalian replicons include those derived from bovine papillomavirus and Epstein-Barr virus. Additionally, the replicon may have two replication systems, thus allowing it to be maintained, for example, in mammalian cells for expression and in a prokaryotic host for cloning and amplification. Examples of such mammalian-bacteria shuttle vectors include pMT2 [Kaufman et al. (1989) Mol. Cell. Biol. 9:946] and pHEBO [Shimizu et al. (1986) Mol. Cell. Biol. 6:1074].

The transformation procedure used depends upon the host to be transformed. Methods for introduction of heterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg. Hep G2), and a number of other cell lines.

ii. Baculovirus Systems

The polynucleotide encoding the protein can also be inserted into a suitable insect expression vector, and is operably linked to the control elements within that vector. Vector construction employs techniques which are known in the art. Generally, the components of the expression system include a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the heterologous gene or genes to be expressed; a wild type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene in to the baculovirus genome); and appropriate insect host cells and growth media.

After inserting the DNA sequence encoding the protein into the transfer vector, the vector and the wild type viral genome are transfected into an insect host cell where the vector and viral genome are allowed to recombine. The packaged recombinant virus is expressed and recombinant plaques are identified and purified. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego Calif. (“MaxBac” kit). These techniques are generally known to those skilled in the art and fully described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987) (hereinafter “Summers and Smith”).

Prior to inserting the DNA sequence encoding the protein into the baculovirus genome, the above described components, comprising a promoter, leader (if desired), coding sequence, and transcription termination sequence, are usually assembled into an intermediate transplacement construct (transfer vector). This may contain a single gene and operably linked regulatory elements; multiple genes, each with its owned set of operably linked regulatory elements; or multiple genes, regulated by the same set of regulatory elements. Intermediate transplacement constructs are often maintained in a replicon, such as an extra-chromosomal element (e.g. plasmids) capable of stable maintenance in a host, such as a bacterium. The replicon will have a replication system, thus allowing it to be maintained in a suitable host for cloning and amplification.

Currently, the most commonly used transfer vector for introducing foreign genes into AcNPV is pAc373. Many other vectors, known to those of skill in the art, have also been designed. These include, for example, pVL985 (which alters the polyhedrin start codon from ATG to ATT, and which introduces a BamHI cloning site 32 basepairs downstream from the ATT; see Luckow and Summers, Virology (1989) 17:31.

The plasmid usually also contains the polyhedrin polyadenylation signal (Miller et al. (1988) Ann. Rev. Microbiol., 42:177) and a prokaryotic ampicillin-resistance (amp) gene and origin of replication for selection and propagation in E. coli.

Baculovirus transfer vectors usually contain a baculovirus promoter. A baculovirus promoter is any DNA sequence capable of binding a baculovirus RNA polymerase and initiating the downstream (5′ to 3′) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A baculovirus transfer vector may also have a second domain called an enhancer, which, if present, is usually distal to the structural gene. Expression may be either regulated or constitutive.

Structural genes, abundantly transcribed at late times in a viral infection cycle, provide particularly useful promoter sequences. Examples include sequences derived from the gene encoding the viral polyhedron protein, Friesen et al., (1986) “The Regulation of Baculovirus Gene Expression,” in: The Molecular Biology of Baculoviruses (ed. Walter Doerfler); EPO Publ. Nos. 127 839 and 155 476; and the gene encoding the p10 protein, Vlak et al., (1988), J. Gen. Virol. 69:765.

DNA encoding suitable signal sequences can be derived from genes for secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene (Carbonell et al. (1988) Gene, 73:409). Alternatively, since the signals for mammalian cell posttranslational modifications (such as signal peptide cleavage, proteolytic cleavage, and phosphorylation) appear to be recognized by insect cells, and the signals required for secretion and nuclear accumulation also appear to be conserved between the invertebrate cells and vertebrate cells, leaders of non-insect origin, such as those derived from genes encoding human α-interferon, Maeda et al., (1985), Nature 315:592; human gastrin-releasing peptide, Lebacq-Verheyden et al., (1988), Molec. Cell. Biol. 8:3129; human IL-2, Smith et al., (1985) Proc. Nat'l Acad. Sci. USA, 82:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; and human glucocerebrosidase, Martin et al. (1988) DNA, 7:99, can also be used to provide for secretion in insects.

A recombinant polypeptide or polyprotein may be expressed intracellularly or, if it is expressed with the proper regulatory sequences, it can be secreted. Good intracellular expression of nonfused foreign proteins usually requires heterologous genes that ideally have a short leader sequence containing suitable translation initiation signals preceding an ATG start signal. If desired, methionine at the N-terminus may be cleaved from the mature protein by in vitro incubation with cyanogen bromide.

Alternatively, recombinant polyproteins or proteins which are not naturally secreted can be secreted from the insect cell by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in insects. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the translocation of the protein into the endoplasmic reticulum.

After insertion of the DNA sequence and/or the gene encoding the expression product precursor of the protein, an insect cell host is co-transformed with the heterologous DNA of the transfer vector and the genomic DNA of wild type baculovirus—usually by co-transfection. The promoter and transcription termination sequence of the construct will usually comprise a 2-5 kb section of the baculovirus genome. Methods for introducing heterologous DNA into the desired site in the baculovirus virus are known in the art. (See Summers and Smith supra; Ju et al. (1987); Smith et al., Mol. Cell. Biol. (1983) 3:2156; and Luckow and Summers (1989)). For example, the insertion can be into a gene such as the polyhedrin gene, by homologous double crossover recombination; insertion can also be into a restriction enzyme site engineered into the desired baculovirus gene. Miller et al., (1989), Bioessays 4:91. The DNA sequence, when cloned in place of the polyhedrin gene in the expression vector, is flanked both 5′ and 3′ by polyhedrin-specific sequences and is positioned downstream of the polyhedrin promoter.

The newly formed baculovirus expression vector is subsequently packaged into an infectious recombinant baculovirus. Homologous recombination occurs at low frequency (between about 1% and about 5%); thus, the majority of the virus produced after cotransfection is still wild-type virus. Therefore, a method is necessary to identify recombinant viruses. An advantage of the expression system is a visual screen allowing recombinant viruses to be distinguished. The polyhedrin protein, which is produced by the native virus, is produced at very high levels in the nuclei of infected cells at late times after viral infection. Accumulated polyhedrin protein forms occlusion bodies that also contain embedded particles. These occlusion bodies, up to 15 μm in size, are highly refractile, giving them a bright shiny appearance that is readily visualized under the light microscope. Cells infected with recombinant viruses lack occlusion bodies. To distinguish recombinant virus from wild-type virus, the transfection supernatant is plagued onto a monolayer of insect cells by techniques known to those skilled in the art. Namely, the plaques are screened under the light microscope for the presence (indicative of wild-type virus) or absence (indicative of recombinant virus) of occlusion bodies. “Current Protocols in Microbiology” Vol. 2 (Ausubel et al. eds) at 16.8 (Supp. 10, 1990); Summers and Smith, supra; Miller et al. (1989).

Recombinant baculovirus expression vectors have been developed for infection into several insect cells. For example, recombinant baculoviruses have been developed for, inter alia: Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni (WO 89/046699; Carbonell et al., (1985) J. Virol. 56:153; Wright (1986) Nature 321:718; Smith et al., (1983) Mol. Cell. Biol. 3:2156; and see generally, Fraser, et al. (1989) In Vitro Cell. Dev. Biol. 25:225).

Cells and cell culture media are commercially available for both direct and fusion expression of heterologous polypeptides in a baculovirus/expression system; cell culture technology is generally known to those skilled in the art. See, eg. Summers and Smith supra.

The modified insect cells may then be grown in an appropriate nutrient medium, which allows for stable maintenance of the plasmid(s) present in the modified insect host. Where the expression product gene is under inducible control, the host may be grown to high density, and expression induced. Alternatively, where expression is constitutive, the product will be continuously expressed into the medium and the nutrient medium must be continuously circulated, while removing the product of interest and augmenting depleted nutrients. The product may be purified by such techniques as chromatography, eg. HPLC, affinity chromatography, ion exchange chromatography, etc.; electrophoresis; density gradient centrifugation; solvent extraction, etc. As appropriate, the product may be further purified, as required, so as to remove substantially any insect proteins which are also present in the medium, so as to provide a product which is at least substantially free of host debris, eg. proteins, lipids and polysaccharides.

In order to obtain protein expression, recombinant host cells derived from the transformants are incubated under conditions which allow expression of the recombinant protein encoding sequence. These conditions will vary, dependent upon the host cell selected. However, the conditions are readily ascertainable to those of ordinary skill in the art, based upon what is known in the art.

iii. Plant Systems

There are many plant cell culture and whole plant genetic expression systems known in the art. Exemplary plant cellular genetic expression systems include those described in patents, such as: U.S. Pat. Nos. 5,693,506; 5,659,122; and 5,608,143. Additional examples of genetic expression in plant cell culture has been described by Zenk, Phytochemistry 30:3861-3863 (1991). Descriptions of plant protein signal peptides may be found in addition to the references described above in Vaulcombe et al., Mol. Gen. Genet. 209:33-40 (1987); Chandler et al., Plant Molecular Biology 3:407-418 (1984); Rogers, J. Biol. Chem. 260:3731-3738 (1985); Rothstein et al., Gene 55:353-356 (1987); Whittier et al., Nucleic Acids Research 15:2515-2535 (1987); Wirsel et al., Molecular Microbiology 3:3-14 (1989); Yu et al., Gene 122:247-253 (1992). A description of the regulation of plant gene expression by the phytohormone, gibberellic acid and secreted enzymes induced by gibberellic acid can be found in R. L. Jones and J. MacMillin, Gibberellins: in: Advanced Plant Physiology, Malcolm B. Wilkins, ed., 1984 Pitman Publishing Limited, London, pp. 21-52. References that describe other metabolically-regulated genes: Sheen, Plant Cell, 2:1027-1038(1990); Maas et al., EMBO J. 9:3447-3452 (1990); Benkel and Hickey, Proc. Natl. Acad. Sci. 84:1337-1339 (1987).

Typically, using techniques known in the art, a desired polynucleotide sequence is inserted into an expression cassette comprising genetic regulatory elements designed for operation in plants. The expression cassette is inserted into a desired expression vector with companion sequences upstream and downstream from the expression cassette suitable for expression in a plant host. The companion sequences will be of plasmid or viral origin and provide necessary characteristics to the vector to permit the vectors to move DNA from an original cloning host, such as bacteria, to the desired plant host. The basic bacterial/plant vector construct will preferably provide a broad host range prokaryote replication origin; a prokaryote selectable marker; and, for Agrobacterium transformations, T DNA sequences for Agrobacterium-mediated transfer to plant chromosomes. Where the heterologous gene is not readily amenable to detection, the construct will preferably also have a selectable marker gene suitable for determining if a plant cell has been transformed. A general review of suitable markers, for example for the members of the grass family, is found in Wilmink and Dons, 1993, Plant Mol. Biol. Reptr, 11(2):165-185.

Sequences suitable for permitting integration of the heterologous sequence into the plant genome are also recommended. These might include transposon sequences and the like for homologous recombination as well as Ti sequences which permit random insertion of a heterologous expression cassette into a plant genome. Suitable prokaryote selectable markers include resistance toward antibiotics such as ampicillin or tetracycline. Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art.

The nucleic acid molecules of the subject invention may be included into an expression cassette for expression of the protein(s) of interest. Usually, there will be only one expression cassette, although two or more are feasible. The recombinant expression cassette will contain in addition to the heterologous protein encoding sequence the following elements, a promoter region, plant 5′ untranslated sequences, initiation codon depending upon whether or not the structural gene comes equipped with one, and a transcription and translation termination sequence. Unique restriction enzyme sites at the 5′ and 3′ ends of the cassette allow for easy insertion into a pre-existing vector.

A heterologous coding sequence may be for any protein relating to the present invention. The sequence encoding the protein of interest will encode a signal peptide which allows processing and translocation of the protein, as appropriate, and will usually lack any sequence which might result in the binding of the desired protein of the invention to a membrane. Since, for the most part, the transcriptional initiation region will be for a gene which is expressed and translocated during germination, by employing the signal peptide which provides for translocation, one may also provide for translocation of the protein of interest. In this way, the protein(s) of interest will be translocated from the cells in which they are expressed and may be efficiently harvested. Typically secretion in seeds are across the aleurone or scutellar epithelium layer into the endosperm of the seed. While it is not required that the protein be secreted from the cells in which the protein is produced, this facilitates the isolation and purification of the recombinant protein.

Since the ultimate expression of the desired gene product will be in a eucaryotic cell it is desirable to determine whether any portion of the cloned gene contains sequences which will be processed out as introns by the host's splicosome machinery. If so, site-directed mutagenesis of the “intron” region may be conducted to prevent losing a portion of the genetic message as a false intron code, Reed and Maniatis, Cell 41:95-105, 1985.

The vector can be microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA. Crossway, Mol. Gen. Genet, 202:179-185, 1985. The genetic material may also be transferred into the plant cell by using polyethylene glycol, Krens, et al., Nature, 296, 72-74, 1982. Another method of introduction of nucleic acid segments is high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface, Klein, et al., Nature, 327, 70-73, 1987 and Knudsen and Muller, 1991, Planta, 185:330-336 teaching particle bombardment of barley endosperm to create transgenic barley. Yet another method of introduction would be fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies, Fraley, et al., Proc. Natl. Acad. Sci. USA, 79, 1859-1863, 1982.

The vector may also be introduced into the plant cells by electroporation. (Fromm et al., Proc. Natl Acad. Sci. USA 82:5824, 1985). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the gene construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus.

All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be transformed by the present invention so that whole plants are recovered which contain the transferred gene. It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables. Some suitable plants include, for example, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Lolium, Zea, Triticum, Sorghum, and Datura.

Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts containing copies of the heterologous gene is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced from the protoplast suspension. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is fully reproducible and repeatable.

In some plant cell culture systems, the desired protein of the invention may be excreted or alternatively, the protein may be extracted from the whole plant. Where the desired protein of the invention is secreted into the medium, it may be collected. Alternatively, the embryos and embryoless-half seeds or other plant tissue may be mechanically disrupted to release any secreted protein between cells and tissues. The mixture may be suspended in a buffer solution to retrieve soluble proteins. Conventional protein isolation and purification methods will be then used to purify the recombinant protein. Parameters of time, temperature pH, oxygen, and volumes will be adjusted through routine methods to optimize expression and recovery of heterologous protein.

iv. Bacterial Systems

Bacterial expression techniques are known in the art. A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A bacterial promoter may also have a second domain called an operator, that may overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene. Constitutive expression may occur in the absence of negative regulatory elements, such as the operator. In addition, positive regulation may be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5′) to the RNA polymerase binding sequence. An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (E. coli) [Raibaud et al. (1984) Annu. Rev. Genet. 18:173]. Regulated expression may therefore be either positive or negative, thereby either enhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) [Chang et al. (1977) Nature 198:1056], and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) [Goeddel et al. (1980) Nuc. Acids Res. 8:4057; Yelverton et al. (1981) Nucl. Acids Res. 9:731; U.S. Pat. No. 4,738,921; EP-A-0036776 and EP-A-0121775]. The g-laotamase (bla) promoter system [Weissmann (1981) “The cloning of interferon and other mistakes.” In Interferon 3 (ed. I. Gresser)], bacteriophage lambda PL [Shimatake et al. (1981) Nature 292:128] and T5 [U.S. Pat. No. 4,689,406] promoter systems also provide useful promoter sequences.

In addition, synthetic promoters which do not occur in nature also function as bacterial promoters. For example, transcription activation sequences of one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433]. For example, the tac promoter is a hybrid trp-lac promoter comprised of both trp promoter and lac operon sequences that is regulated by the lac repressor [Amann et al. (1983) Gene 25:167; de Boer et al. (1983) Proc. Natl. Acad. Sci. 80:21]. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes. The bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system [Studier et al. (1986) J. Mol. Biol. 189:113; Tabor et al. (1985) Proc Natl. Acad. Sci. 82:1074]. In addition, a hybrid promoter can also be comprised of a bacteriophage promoter and an E. coli operator region (EPO-A-0 267 851).

In addition to a functioning promoter sequence, an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes. In E. coli, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon [Shine et al. (1975) Nature 254:34]. The SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3′ and of E. coli 16S rRNA [Steitz et al. (1979) “Genetic signals and nucleotide sequences in messenger RNA.” In Biological Regulation and Development: Gene Expression (ed. R. F. Goldberger)]. To express eukaryotic genes and prokaryotic genes with weak ribosome-binding site [Sambrook et al. (1989) “Expression of cloned genes in Escherichia coli.” In Molecular Cloning: A Laboratory Manual].

A DNA molecule may be expressed intracellularly. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide or by either in vivo on in vitro incubation with a bacterial methionine N-terminal peptidase (EP-A-0 219 237).

Fusion proteins provide an alternative to direct expression. Usually, a DNA sequence encoding the N-terminal portion of an endogenous bacterial protein, or other stable protein, is fused to the 5′ end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the bacteriophage lambda cell gene can be linked at the 5′ terminus of a foreign gene and expressed in bacteria. The resulting fusion protein preferably retains a site for a processing enzyme (factor Xa) to cleave the bacteriophage protein from the foreign gene [Nagai et al. (1984) Nature 309:810]. Fusion proteins can also be made with sequences from the lacZ [Jia et al. (1987) Gene 60:197], trpE [Allen et al. (1987) J. Biotechnol. 5:93; Makoff et al. (1989) J. Gen. Microbiol. 135:11], and Chey [EP-A-0 324 647] genes. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (eg. ubiquitin specific processing-protease) to cleave the ubiquitin from the foreign protein. Through this method, native foreign protein can be isolated [Miller et al. (1989) Bio/Technology 7:698].

Alternatively, foreign proteins can also be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein comprised of a signal peptide sequence fragment that provides for secretion of the foreign protein in bacteria [U.S. Pat. No. 4,336,336]. The signal sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria). Preferably there are processing sites, which can be cleaved either in vivo or in vitro encoded between the signal peptide fragment and the foreign gene.

DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E. coli outer membrane protein gene (ompA) [Masui et al. (1983), in: Experimental Manipulation of Gene Expression; Ghrayeb et al. (1984) EMBO J. 3:2437] and the E. coli alkaline phosphatase signal sequence (phoA) [Oka et al. (1985) Proc. Natl. Acad. Sci. 82:7212]. As an additional example, the signal sequence of the alpha-amylase gene from various Bacillus strains can be used to secrete heterologous proteins from B. subtilis [Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 244 042].

Usually, transcription termination sequences recognized by bacteria are regulatory regions located 3′ to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Transcription termination sequences frequently include DNA sequences of about 50 nucleotides capable of forming stem loop structures that aid in terminating transcription. Examples include transcription termination sequences derived from genes with strong promoters, such as the trp gene in E. coli as well as other biosynthetic genes.

Usually, the above described components, comprising a promoter, signal sequence (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as bacteria. The replicon will have a replication system, thus allowing it to be maintained in a prokaryotic host either for expression or for cloning and amplification. In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150. A host containing a high copy number plasmid will preferably contain at least about 10, and more preferably at least about 20 plasmids. Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host.

Alternatively, the expression constructs can be integrated into the bacterial genome with an integrating vector. Integrating vectors usually contain at least one sequence homologous to the bacterial chromosome that allows the vector to integrate. Integrations appear to result from recombinations between homologous DNA in the vector and the bacterial chromosome. For example, integrating vectors constructed with DNA from various Bacillus strains integrate into the Bacillus chromosome (EP-A-0 127 328). Integrating vectors may also be comprised of bacteriophage or transposon sequences.

Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of bacterial strains that have been transformed. Selectable markers can be expressed in the bacterial host and may include genes which render bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline [Davies et al. (1978) Annu. Rev. Microbiol. 32:469]. Selectable markers may also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.

Alternatively, some of the above described components can be put together in transformation vectors. Transformation vectors are usually comprised of a selectable market that is either maintained in a replicon or developed into an integrating vector, as described above.

Expression and transformation vectors, either extra-chromosomal replicons or integrating vectors, have been developed for transformation into many bacteria. For example, expression vectors have been developed for, inter alia, the following bacteria: Bacillus subtilis [Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541], Escherichia coli [Shimatake et al. (1981) Nature 292:128; Amann et al. (1985) Gene 40:183; Studier et al. (1986) J. Mol. Biol. 189:113; EP-A-0 036 776, EP-A-0 136 829 and EP-A-0 136 907], Streptococcus cremoris [Powell et al. (1988) Appl. Environ. Microbiol. 54:655]; Streptococcus lividans [Powell et al. (1988) Appl. Environ. Microbiol. 54:655], Streptomyces lividans [U.S. Pat. No. 4,745,056].

Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and usually include either the transformation of bacteria treated with CaCl₂ or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation. Transformation procedures usually vary with the bacterial species to be transformed. See eg. [Masson et al. (1989) FEMS Microbiol. Lett. 60:273; Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541, Bacillus], [Miller et al. (1988) Proc. Natl. Acad. Sci. 85:856; Wang et al. (1990) J. Bacteriol. 172:949, Campylobacter], [Cohen et al. (1973) Proc. Natl. Acad. Sci. 69:2110; Dower et al. (1988) Nucleic Acids Res. 16:6127; Kushner (1978) “An improved method for transformation of Escherichia coli with ColE1-derived plasmids. In Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering (eds. H. W. Boyer and S. Nicosia); Mandel et al. (1970) J. Mol. Biol. 53:159; Taketo (1988) Biochim. Biophys. Acta 949:318; Escherichia], [Chassy et al. (1987) FEMS Microbiol. Lett. 44:173 Lactobacillus]; [Fiedler et al. (1988) Anal. Biochem 170:38, Pseudomonas]; [Augustin et al. (1990) FEMS Microbiol. Lett. 66:203, Staphylococcus], [Barany et al. (1980) J. Bacteriol. 144:698; Harlander (1987) “Transformation of Streptococcus lactis by electroporation, in: Streptococcal Genetics (ed. J. Ferretti and R. Curtiss III); Perry et al. (1981) Infect. Immun. 32:1295; Powell et al. (1988) Appl. Environ. Microbiol. 54:655; Somkuti et al. (1987) Proc. 4th Evr. Cong. Biotechnology 1:412, Streptococcus].

v. Yeast Expression

Yeast expression systems are also known to one of ordinary skill in the art. A yeast promoter is any DNA sequence capable of binding yeast RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site (the “TATA Box”) and a transcription initiation site. A yeast promoter may also have a second domain called an upstream activator sequence (UAS), which, if present, is usually distal to the structural gene. The UAS permits regulated (inducible) expression. Constitutive expression occurs in the absence of a UAS. Regulated expression may be either positive or negative, thereby either enhancing or reducing transcription.

Yeast is a fermenting organism with an active metabolic pathway, therefore sequences encoding enzymes in the metabolic pathway provide particularly useful promoter sequences. Examples include alcohol dehydrogenase (ADH) (EP-A-0 284 044), enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK) (EPO-A-0 329 203). The yeast PHO5 gene, encoding acid phosphatase, also provides useful promoter sequences [Myanohara et al. (1983) Proc. Natl. Acad. Sci. USA 80:1].

In addition, synthetic promoters which do not occur in nature also function as yeast promoters. For example, UAS sequences of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter. Examples of such hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region (U.S. Pat. Nos. 4,876,197 and 4,880,734). Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2, GAL4, GAL10, OR PHO5 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (EP-A-0 164 556). Furthermore, a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription. Examples of such promoters include, inter alia, [Cohen et al. (1980) Proc. Natl. Acad. Sci. USA 77:1078; Henikoff et al. (1981) Nature 283:835; Hollenberg et al. (1981) Curr. Topics Microbiol. Immunol. 96:119; Hollenberg et al. (1979) “The Expression of Bacterial Antibiotic Resistance Genes in the Yeast Saccharomyces cerevisiae,” in: Plasmids of Medical, Environmental and Commercial Importance (eds. K. N. Timmis and A. Puhler); Mercerau-Puigalon et al. (1980) Gene 11:163; Panthier et al. (1980) Curr. Genet. 2:109; 1.

A DNA molecule may be expressed intracellularly in yeast. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.

Fusion proteins provide an alternative for yeast expression systems, as well as in mammalian, baculovirus, and bacterial expression systems. Usually, a DNA sequence encoding the N-terminal portion of an endogenous yeast protein, or other stable protein, is fused to the 5′ end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the yeast or human superoxide dismutase (SOD) gene, can be linked at the 5′ terminus of a foreign gene and expressed in yeast. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. See eg. EP-A-0 196 056. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (eg. ubiquitin-specific processing protease) to cleave the ubiquitin from the foreign protein. Through this method, therefore, native foreign protein can be isolated (eg. WO88/024066).

Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provide for secretion in yeast of the foreign protein. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.

DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast invertase gene (EP-A-0 012 873; JPO. 62,096,086) and the A-factor gene (U.S. Pat. No. 4,588,684). Alternatively, leaders of non-yeast origin, such as an interferon leader, exist that also provide for secretion in yeast (EP-A-0 060 057).

A preferred class of secretion leaders are those that employ a fragment of the yeast alpha-factor gene, which contains both a “pre” signal sequence, and a “pro” region. The types of alpha-factor fragments that can be employed include the full-length pre-pro alpha factor leader (about 83 amino acid residues) as well as truncated alpha-factor leaders (usually about 25 to about 50 amino acid residues) (U.S. Pat. Nos. 4,546,083 and 4,870,008; EP-A-0 324 274). Additional leaders employing an alpha-factor leader fragment that provides for secretion include hybrid alpha-factor leaders made with a presequence of a first yeast, but a pro-region from a second yeast alphafactor. (eg. see WO 89/02463.)

Usually, transcription termination sequences recognized by yeast are regulatory regions located 3′ to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminator sequence and other yeast-recognized termination sequences, such as those coding for glycolytic enzymes.

Usually, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as yeast or bacteria. The replicon may have two replication systems, thus allowing it to be maintained, for example, in yeast for expression and in a prokaryotic host for cloning and amplification. Examples of such yeast-bacteria shuttle vectors include YEp24 [Botstein et al. (1979) Gene 8:17-24], pCl/1 [Brake et al. (1984) Proc. Natl. Acad. Sci USA 81:4642-4646], and YRp17 [Stinchcomb et al. (1982) J. Mol. Biol. 158:157]. In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150. A host containing a high copy number plasmid will preferably have at least about 10, and more preferably at least about 20. Enter a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host. See eg. Brake et al., supra.

Alternatively, the expression constructs can be integrated into the yeast genome with an integrating vector. Integrating vectors usually contain at least one sequence homologous to a yeast chromosome that allows the vector to integrate, and preferably contain two homologous sequences flanking the expression construct. Integrations appear to result from recombinations between homologous DNA in the vector and the yeast chromosome [Orr-Weaver et al. (1983) Methods in Enzymol. 101:228-245]. An integrating vector may be directed to a specific locus in yeast by selecting the appropriate homologous sequence for inclusion in the vector. See Orr-Weaver et al., supra. One or more expression construct may integrate, possibly affecting levels of recombinant protein produced [Rine et al. (1983) Proc. Natl. Acad. Sci. USA 80:6750]. The chromosomal sequences included in the vector can occur either as a single segment in the vector, which results in the integration of the entire vector, or two segments homologous to adjacent segments in the chromosome and flanking the expression construct in the vector, which can result in the stable integration of only the expression construct.

Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of yeast strains that have been transformed. Selectable markers may include biosynthetic genes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2, TRP1, and ALG7, and the G418 resistance gene, which confer resistance in yeast cells to tunicamycin and G418, respectively. In addition, a suitable selectable marker may also provide yeast with the ability to grow in the presence of toxic compounds, such as metal. For example, the presence of CUP1 allows yeast to grow in the presence of copper ions [Butt et al. (1987) Microbiol, Rev. 51:351].

Alternatively, some of the above described components can be put together into transformation vectors. Transformation vectors are usually comprised of a selectable marker that is either maintained in a replicon or developed into an integrating vector, as described above.

Expression and transformation vectors, either extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts. For example, expression vectors have been developed for, inter alia, the following yeasts: Candida albicans [Kurtz, et al. (1986) Mol. Cell. Biol. 6:142], Candida maltosa [Kunze, et al. (1985) J. Basic Microbiol. 25:141]. Hansenula polymorpha [Gleeson, et al. (1986) J. Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302], Kluyveromyces fragilis [Das, et al. (1984) J. Bacteriol. 158:1165], Kluyveromyces lactis [De Louvencourt et al. (1983) J. Bacteriol. 154:737; Van den Berg et al. (1990) Bio/Technology 8:135], Pichia guillerimondii [Kunze et al. (1985) J. Basic Microbiol. 25:141], Pichia pastoris [Gregg, et al. (1985) Mol. Cell. Biol. 5:3376; U.S. Pat. Nos. 4,837,148 and 4,929,555], Saccharomyces cerevisiae [Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75:1929; Ito et al. (1983) J. Bacteriol. 153:163], Schizosaccharomyces pombe [Beach and Nurse (1981) Nature 300:706], and Yarrowia lipolytica [Davidow, et al. (1985) Curr. Genet. 10:380471 Gaillardin, et al. (1985) Curr. Genet. 10:49].

Methods of introducing exogenous DNA into yeast hosts are well-known in the art, and usually include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations. Transformation procedures usually vary with the yeast species to be transformed. See eg. [Kurtz et al. (1986) Mol. Cell. Biol. 6:142; Kunze et al. (1985) J. Basic Microbiol. 25:141; Candida]; [Gleeson et al. (1986) J. Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302; Hansenula]; [Das et al. (1984) J. Bacteriol. 158:1165; De Louvencourt et al. (1983) J. Bacteriol. 154:1165; Van den Berg et al. (1990) Bio/Technology 8:135; Kluyveromyces]; [Gregg et al. (1985) Mol. Cell. Biol. 5:3376; Kunze et al. (1985) J. Basic Microbiol. 25:141; U.S. Pat. Nos. 4,837,148 and 4,929,555; Pichia]; [Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75; 1929; Ito et al. (1983) J. Bacteriol. 153:163 Saccharomyces]; [Beach and Nurse (1981) Nature 300:706; Schizosaccharomyces]; [Davidow et al. (1985) Curr. Genet. 10:39; Gaillardin et al. (1985) Curr. Genet. 10:49; Yarrowia].

Antibodies

As used herein, the term “antibody” refers to a polypeptide or group of polypeptides composed of at least one antibody combining site. An “antibody combining site” is the three-dimensional binding space with an internal surface shape and charge distribution complementary to the features of an epitope of an antigen, which allows a binding of the antibody with the antigen. “Antibody” includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, humanised antibodies, altered antibodies, univalent antibodies, Fab proteins, and single domain antibodies.

Antibodies against the proteins of the invention are useful for affinity chromatography, immunoassays, and distinguishing/identifying streptococcus proteins.

Antibodies to the proteins of the invention, both polyclonal and monoclonal, may be prepared by conventional methods. In general, the protein is first used to immunize a suitable animal, preferably a mouse, rat, rabbit or goat. Rabbits and goats are preferred for the preparation of polyclonal sera due to the volume of serum obtainable, and the availability of labeled anti-rabbit and anti-goat antibodies. Immunization is generally performed by mixing or emulsifying the protein in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly). A dose of 50-200 μg/injection is typically sufficient. Immunization is generally boosted 2-6 weeks later with one or more injections of the protein in saline, preferably using Freund's incomplete adjuvant. One may alternatively generate antibodies by in vitro immunization using methods known in the art, which for the purposes of this invention is considered equivalent to in vivo immunization. Polyclonal antisera is obtained by bleeding the immunized animal into a glass or plastic container, incubating the blood at 25° C. for one hour, followed by incubating at 4° C. for 2-18 hours. The serum is recovered by centrifugation (eg. 1,000 g for 10 minutes). About 20-50 ml per bleed may be obtained from rabbits.

Monoclonal antibodies are prepared using the standard method of Kohler & Milstein [Nature (1975) 256:495-96], or a modification thereof. Typically, a mouse or rat is immunized as described above. However, rather than bleeding the animal to extract serum, the spleen (and optionally several large lymph nodes) is removed and dissociated into single cells. If desired, the spleen cells may be screened (after removal of nonspecifically adherent cells) by applying a cell suspension to a plate or well coated with the protein antigen. B-cells expressing membrane-bound immunoglobulin specific for the antigen bind to the plate, and are not rinsed away with the rest of the suspension. Resulting B-cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium (eg. hypoxanthine, aminopterin, thymidine medium, “HAT”). The resulting hybridomas are plated by limiting dilution, and are assayed for production of antibodies which bind specifically to the immunizing antigen (and which do not bind to unrelated antigens). The selected MAb-secreting hybridomas are then cultured either in vitro (eg. in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice).

If desired, the antibodies (whether polyclonal or monoclonal) may be labeled using conventional techniques. Suitable labels include fluorophores, chromophores, radioactive atoms (particularly ³²P and ¹²⁵I), electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are typically detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert 3,3′,5,5′-tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer. “Specific binding partner” refers to a protein capable of binding a ligand molecule with high specificity, as for example in the case of an antigen and a monoclonal antibody specific therefor. Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art. It should be understood that the above description is not meant to categorize the various labels into distinct classes, as the same label may serve in several different modes. For example, ¹²⁵I may serve as a radioactive label or as an electron-dense reagent. HRP may serve as enzyme or as antigen for a MAb. Further, one may combine various labels for desired effect. For example, MAbs and avidin also require labels in the practice of this invention: thus, one might label a MAb with biotin, and detect its presence with avidin labeled with ¹²⁵I, or with an anti-biotin MAb labeled with HRP. Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention.

Pharmaceutical Compositions

Pharmaceutical compositions can comprise either polypeptides, antibodies, or nucleic acid of the invention. The pharmaceutical compositions will comprise a therapeutically effective amount of either polypeptides, antibodies, or polynucleotides of the claimed invention.

The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by routine experimentation and is within the judgement of the clinician.

For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the molecule of the invention in the individual to which it is administered.

A pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.

Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N. J. 1991).

Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier.

Delivery Methods

Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated.

Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (eg. see WO98/20734), needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.

Vaccines

Vaccines according to the invention may either be prophylactic (ie. to prevent infection) or therapeutic (ie. to treat disease after infection).

Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with “pharmaceutically acceptable carriers,” which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents (“adjuvants”). Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, etc. pathogens.

Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59™ (WO90/14837; Chapter 10 in Vaccine Design—the subunit and adjuvant approach (1995) ed. Powell & Newman), containing 5% Squalene, 0.5% TWEEN® 80 (polyoxyethylene sorbitan monoleate), and 0.5% SPAN® 85 (sorbitan trioleate) (optionally containing MTP-PE) formulated into submicron particles using a microfluidizer, (b) SAF, containing 10% Squalane, 0.4% TWEEN® 80, 5% pluronic-blocked polymer L121, and thr-MDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) RIBI™ adjuvant system (RAS), (Bibi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% TWEEN® 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (DETOX™); (2) saponin adjuvants, such as QS21 or STIMULON™ (Cambridge Bioscience, Worcester, Mass.) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes), which ISCOMS may be devoid of additional detergent e.g. WO00/07621; (3) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (4) cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (WO99/44636), etc.), interferons (e.g. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) e.g. GB-2220221, EP-A-0689454; (6) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions e.g. EP-A-0835318, EP-A-0735898, EP-A-0761231; (7) oligonucleotides comprising CpG motifs [Krieg Vaccine 2000, 19, 618-622; Krieg Curr Opin Mol Ther 2001 3:15-24; Roman et al., Nat. Med., 1997, 3, 849-854; Weiner et al., PNAS USA, 1997, 94, 10833-10837; Davis et al., J. Immunol., 1998, 160, 870-876; Chu et al., J. Exp. Med., 1997, 186, 1623-1631; Lipford et al., Eur. J. Immunol., 1997, 27, 2340-2344; Moldoveanu et al., Vaccine, 1988, 16, 1216-1224, Krieg et al., Nature, 1995, 374, 546-549; Klinman et al., PNAS USA, 1996, 93, 2879-2883; Ballas et al., J. Immunol., 1996, 157, 1840-1845; Cowdery et al., J. Immunol., 1996, 156, 4570-4575; Halpern et al., Cell. Immunol., 1996, 167, 72-78; Yamamoto et al., Jpn. J. Cancer Res., 1988, 79, 866-873; Stacey et al., J. Immunol., 1996, 157, 2116-2122; Messina et al., J. Immunol., 1991, 147, 1759-1764; Yi et al., J. Immunol., 1996, 157, 4918-4925; Yi et al., J. Immunol., 1996, 157, 5394-5402; Yi et al., J. Immunol., 1998, 160, 4755-4761; and Yi et al., J. Immunol., 1998, 160, 5898-5906; International patent applications WO96/02555, WO98/16247, WO98/18810, WO98/40100, WO98/55495, WO98/37919 and WO98/52581] i.e. containing at least one CG dinucleotide, with 5-methylcytosine optionally being used in place of cytosine; (8) a polyoxyethylene ether or a polyoxyethylene ester e.g. WO99/52549; (9) a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol (e.g. WO01/21207) or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol (e.g. WO01/21152); (10) an immunostimulatory oligonucleotide (e.g. a CpG oligonucleotide) and a saponin e.g. WO00/62800; (11) an immunostimulant and a particle of metal salt e.g. WO00/23105; (12) a saponin and an oil-in-water emulsion e.g. WO99/11241; (13) a saponin (e.g. QS21)+3dMPL+IL-12 (optionally+a sterol) e.g. WO98/57659; (14) aluminium salts, preferably hydroxide or phosphate, but any other suitable salt may also be used (e.g. hydroxyphosphate, oxyhydroxide, orthophosphate, sulphate etc. [e.g. see chapters 8 & 9 of Powell & Newman]). Mixtures of different aluminium salts may also be used. The salt may take any suitable form (e.g. gel, crystalline, amorphous etc.); (15) other substances that act as immunostimulating agents to enhance the efficacy of the composition. Aluminium salts and/or MF59™ are preferred.

As mentioned above, muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphos-phoryloxy)-ethylamine (MTP-PE), etc.

The immunogenic compositions (eg. the immunising antigen/immuno-gen/polypeptide/protein/nucleic acid, pharmaceutically acceptable carrier, and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.

Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic or immunogenic polypeptides, as well as any other of the above-mentioned components, as needed. By “immunologically effective amount”, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (eg. nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

The immunogenic compositions are conventionally administered parenterally, eg. by injection, either subcutaneously, intramuscularly, or transdermally/transcutaneously (eg. WO98/20734). Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents.

As an alternative to protein-based vaccines, DNA vaccination may be used [eg. Robinson & Torres (1997) Seminars in Immunol 9:271-283; Donnelly et al. (1997) Annu Rev Immunol 15:617-648; later herein].

Gene Delivery Vehicles

Gene therapy vehicles for delivery of constructs including a coding sequence of a therapeutic of the invention, to be delivered to the mammal for expression in the mammal, can be administered either locally or systemically. These constructs can utilize viral or non-viral vector approaches in in vivo or ex vivo modality. Expression of such coding sequence can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence in vivo can be either constitutive or regulated.

The invention includes gene delivery vehicles capable of expressing the contemplated nucleic acid sequences. The gene delivery vehicle is preferably a viral vector and, more preferably, a retroviral, adenoviral, adeno-associated viral (AAV), herpes viral, or alphavirus vector. The viral vector can also be an astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, or togavirus viral vector. See generally, Jolly (1994) Cancer Gene Therapy 1:51-64; Kimura (1994) Human Gene Therapy 5:845-852; Connelly (1995) Human Gene Therapy 6:185-193; and Kaplitt (1994) Nature Genetics 6:148-153.

Retroviral vectors are well known in the art and we contemplate that any retroviral gene therapy vector is employable in the invention, including B, C and D type retroviruses, xenotropic retroviruses (for example, NZB-X1, NZB-X2 and NZB9-1 (see O'Neill (1985) J. Virol. 53:160) polytropic retroviruses eg. MCF and MCF-MLV (see Kelly (1983) J. Virol. 45:291), spumaviruses and lentiviruses. See RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985.

Portions of the retroviral gene therapy vector may be derived from different retroviruses. For example, retrovector LTRs may be derived from a Murine Sarcoma Virus, a tRNA binding site from a Rous Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.

These recombinant retroviral vectors may be used to generate transduction competent retroviral vector particles by introducing them into appropriate packaging cell lines (see U.S. Pat. No. 5,591,624). Retrovirus vectors can be constructed for site-specific integration into host cell DNA by incorporation of a chimeric integrase enzyme into the retroviral particle (see WO96/37626). It is preferable that the recombinant viral vector is a replication defective recombinant virus.

Packaging cell lines suitable for use with the above-described retrovirus vectors are well known in the art, are readily prepared (see WO95/30763 and WO92/05266), and can be used to create producer cell lines (also termed vector cell lines or “VCLs”) for the production of recombinant vector particles. Preferably, the packaging cell lines are made from human parent cells (eg. HT1080 cells) or mink parent cell lines, which eliminates inactivation in human serum.

Preferred retroviruses for the construction of retroviral gene therapy vectors include Avian Leukosis Virus, Bovine Leukemia, Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis Virus and Rous Sarcoma Virus. Particularly preferred Murine Leukemia Viruses include 4070A and 1504A (Hartley and Rowe (1976) J Virol 19:19-25), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC Nol VR-590), Kirsten, Harvey Sarcoma Virus and Rauscher (ATCC No. VR-998) and Moloney Murine Leukemia Virus (ATCC No. VR-190). Such retroviruses may be obtained from depositories or collections such as the American Type Culture Collection (“ATCC”) in Rockville, Md. or isolated from known sources using commonly available techniques.

Exemplary known retroviral gene therapy vectors employable in this invention include those described in patent applications GB2200651, EP0415731, EP0345242, EP0334301, WO89/02468; WO89/05349, WO89/09271, WO90/02806, WO90/07936, WO94/03622, WO93/25698, WO93/25234, WO93/11230, WO93/10218, WO91/02805, WO91/02825, WO95/07994, U.S. Pat. Nos. 5,219,740, 4,405,712, 4,861,719, 4,980,289, 4,777,127, 5,591,624. See also Vile (1993) Cancer Res 53:3860-3864; Vile (1993) Cancer Res 53:962-967; Ram (1993) Cancer Res 53 (1993) 83-88; Takamiya (1992) J Neurosci Res 33:493-503; Baba (1993) J Neurosurg 79:729-735; Mann (1983) Cell 33:153; Cane (1984) Proc Natl Acad Sci 81:6349; and Miller (1990) Human Gene Therapy 1.

Human adenoviral gene therapy vectors are also known in the art and employable in this invention. See, for example, Berkner (1988) Biotechniques 6:616 and Rosenfeld (1991) Science 252:431, and WO93/07283, WO93/06223, and WO93/07282. Exemplary known adenoviral gene therapy vectors employable in this invention include those described in the above referenced documents and in WO94/12649, WO93/03769, WO93/19191, WO94/28938, WO95/11984, WO95/00655, WO95/27071, WO95/29993, WO95/34671, WO96/05320, WO94/08026, WO94/11506, WO93/06223, WO94/24299, WO95/14102, WO95/24297, WO95/02697, WO94/28152, WO94/24299, WO95/09241, WO95/25807, WO95/05835, WO94/18922 and WO95/09654. Alternatively, administration of DNA linked to killed adenovirus as described in Curiel (1992) Hum. Gene Ther. 3:147-154 may be employed. The gene delivery vehicles of the invention also include adenovirus associated virus (AAV) vectors. Leading and preferred examples of such vectors for use in this invention are the AAV-2 based vectors disclosed in Srivastava, WO93/09239. Most preferred AAV vectors comprise the two AAV inverted terminal repeats in which the native D-sequences are modified by substitution of nucleotides, such that at least 5 native nucleotides and up to 18 native nucleotides, preferably at least 10 native nucleotides up to 18 native nucleotides, most preferably 10 native nucleotides are retained and the remaining nucleotides of the D-sequence are deleted or replaced with non-native nucleotides. The native D-sequences of the AAV inverted terminal repeats are sequences of 20 consecutive nucleotides in each AAV inverted terminal repeat (ie. there is one sequence at each end) which are not involved in HP formation. The non-native replacement nucleotide may be any nucleotide other than the nucleotide found in the native D-sequence in the same position. Other employable exemplary AAV vectors are pWP-19, pWN-1, both of which are disclosed in Nahreini (1993) Gene 124:257-262. Another example of such an AAV vector is psub201 (see Samulski (1987) J. Virol. 61:3096). Another exemplary AAV vector is the Double-D ITR vector. Construction of the Double-D ITR vector is disclosed in U.S. Pat. No. 5,478,745. Still other vectors are those disclosed in Carter U.S. Pat. No. 4,797,368 and Muzyczka U.S. Pat. No. 5,139,941, Chartejee U.S. Pat. No. 5,474,935, and Kotin WO94/288157. Yet a further example of an AAV vector employable in this invention is SSV9AFABTKneo, which contains the AFP enhancer and albumin promoter and directs expression predominantly in the liver. Its structure and construction are disclosed in Su (1996) Human Gene Therapy 7:463-470. Additional AAV gene therapy vectors are described in U.S. Pat. Nos. 5,354,678, 5,173,414, 5,139,941, and 5,252,479.

The gene therapy vectors of the invention also include herpes vectors. Leading and preferred examples are herpes simplex virus vectors containing a sequence encoding a thymidine kinase polypeptide such as those disclosed in U.S. Pat. No. 5,288,641 and EP0176170 (Roizman). Additional exemplary herpes simplex virus vectors include HFEM/ICP6-LacZ disclosed in WO95/04139 (Wistar Institute), pHSVlac described in Geller (1988) Science 241:1667-1669 and in WO90/09441 and WO92/07945, HSV Us3::pgC-lacZ described in Fink (1992) Human Gene Therapy 3:11-19 and HSV 7134, 2 RH 105 and GAL4 described in EP 0453242 (Breakefield), and those deposited with the ATCC with accession numbers VR-977 and VR-260.

Also contemplated are alpha virus gene therapy vectors that can be employed in this invention. Preferred alpha virus vectors are Sindbis viruses vectors. Togaviruses, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532), and those described in U.S. Pat. Nos. 5,091,309, 5,217,879, and WO92/10578. More particularly, those alpha virus vectors described in U.S. Ser. No. 08/405,627, filed Mar. 15, 1995, WO94/21792, WO92/10578, WO95/07994, U.S. Pat. Nos. 5,091,309 and 5,217,879 are employable. Such alpha viruses may be obtained from depositories or collections such as the ATCC in Rockville, Md. or isolated from known sources using commonly available techniques. Preferably, alphavirus vectors with reduced cytotoxicity are used (see U.S. Ser. No. 08/679,640).

DNA vector systems such as eukaryotic layered expression systems are also useful for expressing the nucleic acids of the invention. See WO95/07994 for a detailed description of eukaryotic layered expression systems. Preferably, the eukaryotic layered expression systems of the invention are derived from alphavirus vectors and most preferably from Sindbis viral vectors.

Other viral vectors suitable for use in the present invention include those derived from poliovirus, for example ATCC VR-58 and those described in Evans, Nature 339 (1989) 385 and Sabin (1973) J. Biol. Standardization 1:115; rhinovirus, for example ATCC VR-1110 and those described in Arnold (1990) J Cell Biochem L401; pox viruses such as canary pox virus or vaccinia virus, for example ATCC VR-111 and ATCC VR-2010 and those described in Fisher-Hoch (1989) Proc Natl Acad Sci 86:317; Flexner (1989) Ann NY Acad Sci 569:86, Flexner (1990) Vaccine 8:17; in U.S. Pat. Nos. 4,603,112 and 4,769,330 and WO89/01973; SV40 virus, for example ATCC VR-305 and those described in Mulligan (1979) Nature 277:108 and Madzak (1992) J Gen Virol 73:1533; influenza virus, for example ATCC VR-797 and recombinant influenza viruses made employing reverse genetics techniques as described in U.S. Pat. No. 5,166,057 and in Enami (1990) Proc Natl Acad Sci 87:3802-3805; Enami & Palese (1991) J Virol 65:2711-2713 and Luytjes (1989) Cell 59:110, (see also McMichael (1983) NET Med 309:13, and Yap (1978) Nature 273:238 and Nature (1979) 277:108); human immunodeficiency virus as described in EP-0386882 and in Buchschacher (1992) J. Virol. 66:2731; measles virus, for example ATCC VR-67 and VR-1247 and those described in EP-0440219; Aura virus, for example ATCC VR-368; Bebaru virus, for example ATCC VR-600 and ATCC VR-1240; Cabassou virus, for example ATCC VR-922; Chikungunya virus, for example ATCC VR-64 and ATCC VR-1241; Fort Morgan Virus, for example ATCC VR-924; Getah virus, for example ATCC VR-369 and ATCC VR-1243; Kyzylagach virus, for example ATCC VR-927; Mayaro virus, for example ATCC VR-66; Mucambo virus, for example ATCC VR-580 and ATCC VR-1244; Ndumu virus, for example ATCC VR-371; Pixuna virus, for example ATCC VR-372 and ATCC VR-1245; Tonate virus, for example ATCC VR-925; Triniti virus, for example ATCC VR-469; Una virus, for example ATCC VR-374; Whataroa virus, for example ATCC VR-926; Y-62-33 virus, for example ATCC VR-375; O'Nyong virus, Eastern encephalitis virus, for example ATCC VR-65 and ATCC VR-1242; Western encephalitis virus, for example ATCC VR-70, ATCC VR-1251, ATCC VR-622 and ATCC VR-1252; and coronavirus, for example ATCC VR-740 and those described in Hamre (1966) Proc Soc Exp Biol Med 121:190.

Delivery of the compositions of this invention into cells is not limited to the above mentioned viral vectors. Other delivery methods and media may be employed such as, for example, nucleic acid expression vectors, polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example see U.S. Ser. No. 08/366,787, filed Dec. 30, 1994 and Curiel (1992) Hum Gene Ther 3:147-154 ligand linked DNA, for example see Wu (1989) J Biol Chem 264:16985-16987, eucaryotic cell delivery vehicles cells, for example see U.S. Ser. No. 08/240,030, filed May 9, 1994, and U.S. Ser. No. 08/404,796, deposition of photopolymerized hydrogel materials, hand-held gene transfer particle gun, as described in U.S. Pat. No. 5,149,655, ionizing radiation as described in U.S. Pat. No. 5,206,152 and in WO92/11033, nucleic charge neutralization or fusion with cell membranes. Additional approaches are described in Philip (1994) Mol Cell Biol 14:2411-2418 and in Woffendin (1994) Proc Natl Acad Sci 91:1581-1585.

Particle mediated gene transfer may be employed, for example see U.S. Ser. No. 60/023,867. Briefly, the sequence can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, as described in Wu & Wu (1987) J. Biol. Chem. 262:4429-4432, insulin as described in Hucked (1990) Biochem Phannacol 40:253-263, galactose as described in Plank (1992) Bioconjugate Chem 3:533-539, lactose or transferrin.

Naked DNA may also be employed. Exemplary naked DNA introduction methods are described in WO 90/11092 and U.S. Pat. No. 5,580,859. Uptake efficiency may be improved using biodegradable latex beads. DNA coated latex beads are efficiently transported into cells after endocytosis initiation by the beads. The method may be improved further by treatment of the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm.

Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120, WO95/13796, WO94/23697, WO91/14445 and EP-524,968. As described in U.S. Ser. No. 60/023,867, on non-viral delivery, the nucleic acid sequences encoding a polypeptide can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then be incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, insulin, galactose, lactose, or transferrin. Other delivery systems include the use of liposomes to encapsulate DNA comprising the gene under the control of a variety of tissue-specific or ubiquitously-active promoters. Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al (1994) Proc. Natl. Acad. Sci. USA 91(24):11581-11585. Moreover, the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials. Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun, as described in U.S. Pat. No. 5,149,655; use of ionizing radiation for activating transferred gene, as described in U.S. Pat. No. 5,206,152 and WO92/11033

Exemplary liposome and polycationic gene delivery vehicles are those described in U.S. Pat. Nos. 5,422,120 and 4,762,915; in WO 95/13796; WO94/23697; and WO91/14445; in EP-0524968; and in Stryer, Biochemistry, pages 236-240 (1975) W.H. Freeman, San Francisco; Szoka (1980) Biochem Biophys Acta 600:1; Bayer (1979) Biochem Biophys Acta 550:464; Rivnay (1987) Meth Enzymol 149:119; Wang (1987) Proc Natl Acad Sci 84:7851; Plant (1989) Anal Biochem 176:420.

A polynucleotide composition can comprises therapeutically effective amount of a gene therapy vehicle, as the term is defined above. For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.

Delivery Methods

Once formulated, the polynucleotide compositions of the invention can be administered (1) directly to the subject; (2) delivered ex vivo, to cells derived from the subject; or (3) in vitro for expression of recombinant proteins. The subjects to be treated can be mammals or birds. Also, human subjects can be treated.

Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (eg. see WO98/20734), needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.

Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in eg. WO93/14778. Examples of cells useful in ex vivo applications include, for example, stem cells, particularly hematopoetic, lymph cells, macrophages, dendritic cells, or tumor cells.

Generally, delivery of nucleic acids for both ex vivo and in vitro applications can be accomplished by the following procedures, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.

Polynucleotide and Polypeptide Pharmaceutical Compositions

In addition to the pharmaceutically acceptable carriers and salts described above, the following additional agents can be used with polynucleotide and/or polypeptide compositions.

A. Polypeptides

One example are polypeptides which include, without limitation: asioloorosomucoid (ASOR); transferrin; asialoglycoproteins; antibodies; antibody fragments; ferritin; interleukins; interferons, granulocyte, macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), stem cell factor and erythropoietin. Viral antigens, such as envelope proteins, can also be used. Also, proteins from other invasive organisms, such as the 17 amino acid peptide from the circumsporozoite protein of plasmodium falciparum known as RII.

B. Hormones, Vitamins, Etc.

Other groups that can be included are, for example: hormones, steroids, androgens, estrogens, thyroid hormone, or vitamins, folic acid.

C. Polyalkylenes, Polysaccharides, Etc.

Also, polyalkylene glycol can be included with the desired polynucleotides/polypeptides. In a preferred embodiment, the polyalkylene glycol is polyethlylene glycol. In addition, mono-, di-, or polysaccharides can be included. In a preferred embodiment of this aspect, the polysaccharide is dextran or DEAE-dextran. Also, chitosan and poly(lactide-co-glycolide)

D. Lipids, and Liposomes

The desired polynucleotide/polypeptide can also be encapsulated in lipids or packaged in liposomes prior to delivery to the subject or to cells derived therefrom.

Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid. The ratio of condensed polynucleotide to lipid preparation can vary but will generally be around 1:1 (mg DNA:micromoles lipid), or more of lipid. For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight (1991) Biochim. Biophys. Acta. 1097:1-17; Straubinger (1983) Meth. Enzymol. 101:512-527.

Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner (1987) Proc. Natl. Acad. Sci. USA 84:7413-7416); mRNA (Malone (1989) Proc. Natl. Acad. Sci. USA 86:6077-6081); and purified transcription factors (Debs (1990) J. Biol. Chem. 265:10189-10192), in functional form.

Cationic liposomes are readily available. For example, N-[1-2,3-dioleyloxy)propyl]-N,N,N-triethyl-ammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner supra). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, eg. Szoka (1978) Proc. Natl. Acad. Sci. USA 75:4194-4198; WO90/11092 for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.

Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

The liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). The various liposome-nucleic acid complexes are prepared using methods known in the art. See eg. Straubinger (1983) Meth. Immunol. 101:512-527; Szoka (1978) Proc. Natl. Acad. Sci. USA 75:4194-4198; Papahadjopoulos (1975) Biochim. Biophys. Acta 394:483; Wilson (1979) Cell 17:77); Deamer & Bangham (1976) Biochim. Biophys. Acta 443:629; Ostro (1977) Biochem. Biophys. Res. Commun. 76:836; Fraley (1979) Proc. Natl. Acad. Sci. USA 76:3348); Enoch & Strittmatter (1979) Proc. Natl. Acad. Sci. USA 76:145; Fraley (1980) J. Biol. Chem. (1980) 255:10431; Szoka & Papahadjopoulos (1978) Proc. Natl. Acad. Sci. USA 75:145; and Schaefer-Ridder (1982) Science 215:166.

E. Lipoproteins

In addition, lipoproteins can be included with the polynucleotide/polypeptide to be delivered. Examples of lipoproteins to be utilized include: chylomicrons, HDL, IDL, LDL, and VLDL. Mutants, fragments, or fusions of these proteins can also be used. Also, modifications of naturally occurring lipoproteins can be used, such as acetylated LDL. These lipoproteins can target the delivery of polynucleotides to cells expressing lipoprotein receptors. Preferably, if lipoproteins are including with the polynucleotide to be delivered, no other targeting ligand is included in the composition.

Naturally occurring lipoproteins comprise a lipid and a protein portion. The protein portion are known as apoproteins. At the present, apoproteins A, B, C, D, and E have been isolated and identified. At least two of these contain several proteins, designated by Roman numerals, AI, AII, AIV; CI, CII, CIII.

A lipoprotein can comprise more than one apoprotein. For example, naturally occurring chylomicrons comprises of A, B, C & E, over time these lipoproteins lose A and acquire C & E. VLDL comprises A, B, C & E apoproteins, LDL comprises apoprotein B; and HDL comprises apoproteins A, C, & E.

The amino acid of these apoproteins are known and are described in, for example, Breslow (1985) Annu Rev. Biochem 54:699; Law (1986) Adv. Exp Med. Biol. 151:162; Chen (1986) J Biol Chem 261:12918; Kane (1980) Proc Natl Acad Sci USA 77:2465; and Utermann (1984) Hum Genet 65:232.

Lipoproteins contain a variety of lipids including, triglycerides, cholesterol (free and esters), and phospholipids. The composition of the lipids varies in naturally occurring lipoproteins. For example, chylomicrons comprise mainly triglycerides. A more detailed description of the lipid content of naturally occurring lipoproteins can be found, for example, in Meth. Enzymol. 128 (1986). The composition of the lipids is chosen to aid in conformation of the apoprotein for receptor binding activity. The composition of lipids can also be chosen to facilitate hydrophobic interaction and association with the polynucleotide binding molecule.

Naturally occurring lipoproteins can be isolated from serum by ultracentrifugation, for instance. Such methods are described in Meth. Enzymol. (supra); Pitas (1980) J. Biochem. 255:5454-5460 and Mahey (1979) J Clin. Invest 64:743-750. Lipoproteins can also be produced by in vitro or recombinant methods by expression of the apoprotein genes in a desired host cell. See, for example, Atkinson (1986) Annu Rev Biophys Chem 15:403 and Radding (1958) Biochim Biophys Acta 30: 443. Lipoproteins can also be purchased from commercial suppliers, such as Biomedical Technologies, Inc., Stoughton, Mass., USA. Further description of lipoproteins can be found in WO98/06437.

F. Polycationic Agents

Polycationic agents can be included, with or without lipoprotein, in a composition with the desired polynucleotide/polypeptide to be delivered.

Polycationic agents, typically, exhibit a net positive charge at physiological relevant pH and are capable of neutralizing the electrical charge of nucleic acids to facilitate delivery to a desired location. These agents have both in vitro, ex vivo, and in vivo applications. Polycationic agents can be used to deliver nucleic acids to a living subject either intramuscularly, subcutaneously, etc.

The following are examples of useful polypeptides as polycationic agents: polylysine, polyarginine, polyornithine, and protamine. Other examples include histones, protamines, human serum albumin, DNA binding proteins, non-histone chromosomal proteins, coat proteins from DNA viruses, such as (X174, transcriptional factors also contain domains that bind DNA and therefore may be useful as nucleic aid condensing agents. Briefly, transcriptional factors such as C/CEBP, c-jun, c-fos, AP-1, AP-2, AP-3, CPF, Prot-1, Sp-1, Oct-1, Oct-2, CREP, and TFIID contain basic domains that bind DNA sequences.

Organic polycationic agents include: spermine, spermidine, and purtrescine.

The dimensions and of the physical properties of a polycationic agent can be extrapolated from the list above, to construct other polypeptide polycationic agents or to produce synthetic polycationic agents.

Synthetic polycationic agents which are useful include, for example, DEAE-dextran, polybrene. Lipofectin™, and LIPOFECTAMINE™ are monomers that form polycationic complexes when combined with polynucleotides/polypeptides.

Immunodiagnostic Assays

Streptococcus antigens of the invention can be used in immunoassays to detect antibody levels (or, conversely, anti-streptococcus antibodies can be used to detect antigen levels). Immunoassays based on well defined, recombinant antigens can be developed to replace invasive diagnostics methods. Antibodies to streptococcus proteins within biological samples, including for example, blood or serum samples, can be detected. Design of the immunoassays is subject to a great deal of variation, and a variety of these are known in the art. Protocols for the immunoassay may be based, for example, upon competition, or direct reaction, or sandwich type assays. Protocols may also, for example, use solid supports, or may be by immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the probe are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.

Kits suitable for immunodiagnosis and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including the compositions of the invention, in suitable containers, along with the remaining reagents and materials (for example, suitable buffers, salt solutions, etc.) required for the conduct of the assay, as well as suitable set of assay instructions.

Nucleic Acid Hybridisation

“Hybridization” refers to the association of two nucleic acid sequences to one another by hydrogen bonding. Typically, one sequence will be fixed to a solid support and the other will be free in solution. Then, the two sequences will be placed in contact with one another under conditions that favor hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase sequence to the solid support (Denhardt's reagent or BLOTTO); concentration of the sequences; use of compounds to increase the rate of association of sequences (dextran sulfate or polyethylene glycol); and the stringency of the washing conditions following hybridization. See Sambrook et al. [supra] Volume 2, chapter 9, pages 9.47 to 9.57.

“Stringency” refers to conditions in a hybridization reaction that favor association of very similar sequences over sequences that differ. For example, the combination of temperature and salt concentration should be chosen that is approximately 120 to 200° C. below the calculated Tm of the hybrid under study. The temperature and salt conditions can often be determined empirically in preliminary experiments in which samples of genomic DNA immobilized on filters are hybridized to the sequence of interest and then washed under conditions of different stringencies. See Sambrook et al. at page 9.50.

Variables to consider when performing, for example, a Southern blot are (1) the complexity of the DNA being blotted and (2) the homology between the probe and the sequences being detected. The total amount of the fragment(s) to be studied can vary a magnitude of 10, from 0.1 to 1 μg for a plasmid or phage digest to 10⁻⁹ to 10⁻⁸ g for a single copy gene in a highly complex eukaryotic genome. For lower complexity polynucleotides, substantially shorter blotting, hybridization, and exposure times, a smaller amount of starting polynucleotides, and lower specific activity of probes can be used. For example, a single-copy yeast gene can be detected with an exposure time of only 1 hour starting with 1 μg of yeast DNA, blotting for two hours, and hybridizing for 4-8 hours with a probe of 10⁸ cpm/μg. For a single-copy mammalian gene a conservative approach would start with 10 μg of DNA, blot overnight, and hybridize overnight in the presence of 10% dextran sulfate using a probe of greater than 10⁸ cpm/μg, resulting in an exposure time of ˜24 hours.

Several factors can affect the melting temperature (Tm) of a DNA-DNA hybrid between the probe and the fragment of interest, and consequently, the appropriate conditions for hybridization and washing. In many cases the probe is not 100% homologous to the fragment. Other commonly encountered variables include the length and total G+C content of the hybridizing sequences and the ionic strength and formamide content of the hybridization buffer. The effects of all of these factors can be approximated by a single equation: Tm=81+16.6(log₁₀ Ci)+0.4[%(G+C)]−0.6(% formamide)−600/n−1.5(% mismatch).

where Ci is the salt concentration (monovalent ions) and n is the length of the hybrid in base pairs (slightly modified from Meinkoth & Wahl (1984) Anal. Biochem. 138: 267-284).

In designing a hybridization experiment, some factors affecting nucleic acid hybridization can be conveniently altered. The temperature of the hybridization and washes and the salt concentration during the washes are the simplest to adjust. As the temperature of the hybridization increases (ie. stringency), it becomes less likely for hybridization to occur between strands that are nonhomologous, and as a result, background decreases. If the radiolabeled probe is not completely homologous with the immobilized fragment (as is frequently the case in gene family and interspecies hybridization experiments), the hybridization temperature must be reduced, and background will increase. The temperature of the washes affects the intensity of the hybridizing band and the degree of background in a similar manner. The stringency of the washes is also increased with decreasing salt concentrations.

In general, convenient hybridization temperatures in the presence of 50% formamide are 42° C. for a probe with is 95% to 100% homologous to the target fragment, 37° C. for 90% to 95% homology, and 32° C. for 85% to 90% homology. For lower homologies, formamide content should be lowered and temperature adjusted accordingly, using the equation above. If the homology between the probe and the target fragment are not known, the simplest approach is to start with both hybridization and wash conditions which are nonstringent. If non-specific bands or high background are observed after autoradiography, the filter can be washed at high stringency and reexposed. If the time required for exposure makes this approach impractical, several hybridization and/or washing stringencies should be tested in parallel.

Nucleic Acid Probe Assays

Methods such as PCR, branched DNA probe assays, or blotting techniques utilizing nucleic acid probes according to the invention can determine the presence of cDNA or mRNA. A probe is said to “hybridize” with a sequence of the invention if it can form a duplex or double stranded complex, which is stable enough to be detected.

The nucleic acid probes will hybridize to the streptococcus nucleotide sequences of the invention (including both sense and antisense strands). Though many different nucleotide sequences will encode the amino acid sequence, the native streptococcus sequence is preferred because it is the actual sequence present in cells. mRNA represents a coding sequence and so a probe should be complementary to the coding sequence; single-stranded cDNA is complementary to mRNA, and so a cDNA probe should be complementary to the non-coding sequence.

The probe sequence need not be identical to the streptococcus sequence (or its complement)—some variation in the sequence and length can lead to increased assay sensitivity if the nucleic acid probe can form a duplex with target nucleotides, which can be detected. Also, the nucleic acid probe can include additional nucleotides to stabilize the formed duplex. Additional streptococcus sequence may also be helpful as a label to detect the formed duplex. For example, a non-complementary nucleotide sequence may be attached to the 5′ end of the probe, with the remainder of the probe sequence being complementary to a streptococcus sequence. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the a streptococcus sequence in order to hybridize therewith and thereby form a duplex which can be detected.

The exact length and sequence of the probe will depend on the hybridization conditions (e.g. temperature, salt condition etc.). For example, for diagnostic applications, depending on the complexity of the analyte sequence, the nucleic acid probe typically contains at least 10-20 nucleotides, preferably 15-25, and more preferably at least 30 nucleotides, although it may be shorter than this. Short primers generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.

Probes may be produced by synthetic procedures, such as the triester method of Matteucci et al. [J. Am. Chem. Soc. (1981) 103:3185], or according to Urdea et al. [Proc. Natl. Acad. Sci. USA (1983) 80: 7461], or using commercially available automated oligonucleotide synthesizers.

The chemical nature of the probe can be selected according to preference. For certain applications, DNA or RNA are appropriate. For other applications, modifications may be incorporated eg. backbone modifications, such as phosphorothioates or methylphosphonates, can be used to increase in vivo half-life, alter RNA affinity, increase nuclease resistance etc. [eg. see Agrawal & Iyer (1995) Curr Opin Biotechnol 6:12-19; Agrawal (1996) TIBTECH 14:376-387]; analogues such as peptide nucleic acids may also be used [eg. see Corey (1997) TIBTECH 15:224-229; Buchardt et al. (1993) TIBTECH 11:384-386].

Alternatively, the polymerase chain reaction (PCR) is another well-known means for detecting small amounts of target nucleic acid. The assay is described in Mullis et al. [Meth. Enzymol. (1987) 155:335-350] & U.S. Pat. Nos. 4,683,195 & 4,683,202. Two “primer” nucleotides hybridize with the target nucleic acids and are used to prime the reaction. The primers can comprise sequence that does not hybridize to the sequence of the amplification target (or its complement) to aid with duplex stability or, for example, to incorporate a convenient restriction site. Typically, such sequence will flank the desired streptococcus sequence.

A thermostable polymerase creates copies of target nucleic acids from the primers using the original target nucleic acids as a template. After a threshold amount of target nucleic acids are generated by the polymerase, they can be detected by more traditional methods, such as Southern blots. When using the Southern blot method, the labelled probe will hybridize to the streptococcus sequence (or its complement).

Also, mRNA or cDNA can be detected by traditional blotting techniques described in Sambrook et al [supra]. mRNA, or cDNA generated from mRNA using a polymerase enzyme, can be purified and separated using gel electrophoresis. The nucleic acids on the gel are then blotted onto a solid support, such as nitrocellulose. The solid support is exposed to a labelled probe and then washed to remove any unhybridized probe. Next, the duplexes containing the labeled probe are detected. Typically, the probe is labelled with a radioactive moiety.

Example

The following example describes nucleic acid sequences which have been identified in Streptococcus, along with their inferred translation products. The example is generally in the following format:

-   -   a nucleotide sequence which has been identified in Streptococcus     -   the inferred translation product of this sequence     -   a computer analysis (e.g. PSORT output) of the translation         product, indicating antigenicity.

The example describes nucleotide sequences from S. agalactiae. The specific strain which was sequenced was from serotype V, and is a clinical strain isolated in Italy which expresses the R antigen (ISS/Rome/Italy collection, strain.2603 V/R). The corresponding sequences from S. pyogenes are also given. Where GBS and GAS show homology in this way, there is conservation between species which suggests an essential function and also gives good cross-species reactivity.

The example includes details of homology to sequences in the public databases. Proteins that are similar in sequence are generally similar in both structure and function, and the homology often indicates a common evolutionary origin. Comparison with sequences of proteins of known function is widely used as a guide for the assignment of putative protein function to a new sequence and has proved particularly useful in whole-genome analyses.

Various tests can be used to assess the in vivo immunogenicity of the proteins identified in the example. For example, the proteins can be expressed recombinantly and used to screen patient sera by immunoblot. A positive reaction between the protein and patient serum indicates that the patient has previously mounted an immune response to the protein in question i.e. the protein is an immunogen. This method can also be used to identify immunodominant proteins. The mouse model used in the example can also be used.

The recombinant protein can also be conveniently used to prepare antibodies e.g. in a mouse. These can be used for direct confirmation that a protein is located on the cell-surface. Labelled antibody (e.g. fluorescent labelling for FACS) can be incubated with intact bacteria and the presence of label on the bacterial surface confirms the location of the protein.

For many GBS proteins, the following data are given:

-   -   SDS-PAGE analysis of total recombinant E. coli cell extracts for         GBS protein expression     -   SDS-PAGE analysis after the protein purification     -   Western-blot analysis of GBS total cell extract using antisera         raised against recombinant proteins     -   FACS and ELISA analysis against GBS using antisera raise against         recombinant proteins     -   Results of the in vivo passive protection assay

Details of experimental techniques used are presented below:

Sequence Analysis

Open reading frames (ORFs) within nucleotide sequences were predicted using the GLIMMER program [Salzberg et al. (1998) Nucleic Acids Res 26:544-8]. Where necessary, start codons were modified and corrected manually on the basis of the presence of ribosome-binding sites and promoter regions on the upstream DNA sequence.

ORFs were then screened against the non-redundant protein databases using the programs BLASTp [Altschul et al. (1990) J. Mol. Biol. 215:403-410] and PRAZE, a modification of the Smith-Waterman algorithm [Smith & Waterman (1981) J Mol Biol 147:195-7; see Fleischmann et al (1995) Science 269:496-512].

Leader peptides within the ORFs were located using three different approaches: (i) PSORT [Nakai (1991) Bull. Inst. Chem. Res., Kyoto Univ. 69:269-291; Horton & Nakai (1996) Intellig. Syst. Mol. Biol. 4:109-115; Horton & Nakai (1997) Intellig. Syst. Mol. Biol. 5:147-152]; (ii) SignalP [Nielsen & Krogh (1998) in Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology (ISMB 6), AAAI Press, Menlo Park, Calif., pp. 122-130; Nielsen et al. (1999) Protein Engineering 12:3-9; Nielsen et al. (1997). Int. J. Neural Sys. 8:581-599]; and (iii) visual inspection of the ORF sequences. Where a signal sequences is given a “possible site” value, the value represents the C-terminus residue of the signal peptide e.g. a “possible site” of 26 means that the signal sequence consists of amino acids 1-26.

Lipoprotein-specific signal peptides were located using three different approaches: (i) PSORT [see above]; (ii) the “prokaryotic membrane lipoprotein lipid attachment site” PROSITE motif [Hofmann et al. (1999) Nucleic Acids Res. 27:215-219; Bucher & Bairoch (1994) in Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology (ISMB-94), AAAI Press, pages 53-61]; and (iii) the FINDPATTERNS program available in the GCG Wisconsin Package, using the pattern (M,L,V)×{9,35}LxxCx.

Transmembrane domains were located using two approaches: (i) PSORT [see above]; (ii) TopPred [von Heijne (1992) J. Mol. Biol. 225:487-494].

LPXTG motifs, characteristic of cell-wall attached proteins in Gram-positive bacteria [Fischetti et al. (1990) Mol Microbiol 4:1603-5] were located with FINDPATTERNS using the pattern (L,I,V,M,Y,F)Px(T,A,S,G)(G,N,S,T,A,L).

RGD motifs, characteristic of cell-adhesion molecules [D'Souza et al. (1991) Trends Biochem Sci 16:246-50] were located using FINDPATTERNS.

Enzymes belonging to the glycolytic pathway were also selected as antigens, because these have been found experimentally expressed on the surface of Streptococci [e.g. Pancholi & Fischetti (1992) J Exp Med 176:415-26; Pancholi & Fischetti (1998) J Biol Chem 273:14503-15].

Cloning, Expression and Purification of Proteins

GBS genes were cloned to facilitate expression in E. coli as two different types of fusion proteins:

-   -   a) proteins having a hexa-histidine tag at the amino-terminus         (His-gbs)     -   b) proteins having a GST fusion partner at the amino-terminus         (Gst-gbs)

Cloning was performed using the Gateway™ technology (Life Technologies), which is based on the site-specific recombination reactions that mediate integration and excision of phage lambda into and from the E. coli genome. A single cloning experiment included the following steps:

-   -   1—Amplification of GBS chromosomal DNA to obtain a PCR product         coding for a single ORF flanked by attB recombination sites.     -   2—Insertion of the PCR product into a pDONR vector (containing         attP sites) through a BP reaction (attB×attP sites). This         reaction gives a so called ‘pEntry’ vector, which now contains         attL sites flanking the insert.     -   3—Insertion of the GBS gene into E. coli expression vectors         (pDestination vectors, containing attR sites) through a LR         reaction between pEntry and pDestination plasmids (attL×attR         sites).

A) Chromosomal DNA Preparation

For chromosomal DNA preparation, GBS strain 2603 V/R (Istituto Superiore Sanità, Rome) was grown to exponential phase in 2 liters TH Broth (Difco) at 37° C., harvested by centrifugation, and dissolved in 40 ml TES (50 mM Tris pH 8, 5 mM EDTA pH 8, 20% sucrose). After addition of 2.5 ml lysozyme solution (25 mg/ml in TES) and 0.5 ml mutanolysin (Sigma M-9901, 25000 U/ml in H₂O), the suspension was incubated at 37° C. for 1 hour. 1 ml RNase (20 mg/ml) and 0.1 ml proteinase K (20 mg/ml) were added and incubation was continued for 30 min. at 37° C.

Cell lysis was obtained by adding 5 ml sarkosyl solution (10% N-laurylsarcosine in 250 mM EDTA pH 8.0), and incubating 1 hour at 37° C. with frequent inversion. After sequential extraction with phenol, phenol-chloroform and chloroform, DNA was precipitated with 0.3M sodium acetate pH 5.2 and 2 volumes of absolute ethanol. The DNA pellet was rinsed with 70% ethanol and dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). DNA concentration was evaluated by OD₂₆₀.

B) Oligonucleotide Design

Synthetic oligonucleotide primers were designed on the basis of the coding sequence of each ORF. The aim was to express the protein's extracellular region. Accordingly, predicted signal peptides were omitted (by deducing the 5′ end amplification primer sequence immediately downstream from the predicted leader sequence) and C-terminal cell-wall anchoring regions were removed (e.g. LPXTG motifs and downstream amino acids). Where additional nucleotides have been deleted, this is indicated by the suffix ‘d’ (e.g. ‘GBS352d’). Conversely, a suffix ‘L’ refers to expression without these deletions. Deletions of C- or N-terminal residues were also sometimes made, as indicated by a ‘C’ or ‘N’ suffix.

The amino acid sequences of the expressed GBS proteins (including ‘d’ and ‘L’ forms etc.) are definitively defined by the sequences of the oligonucleotide primers.

5′ tails of forward primers and 3′ tails of reverse primers included attB1 and attB2 sites respectively:

Forward Primers:

5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTCT-ORF in frame-3′ (nucleotides 1-31 of SEQ ID NO:11313; the TCT sequence preceding the ORF was omitted when the ORF's first coding triplet began with T).

Reverse Primers:

5′-GGGGACCACTTTGTACAAGAAAGCTGGGTT-ORF reverse complement-3′ (nucleotides 1-30 of SEQ ID NO:11838).

The primers for GBS317 are thus:

Fwd: (SEQ ID NO: 11313) 5′-ggggacaagtttgtacaaaaaagcaggctctaataagccatattca atag-3′ Rev: (SEQ ID NO: 11838) 5′-ggggaccactttgtacaagaaagctgggttatcttctcctaactta ccc-3′

The number of nucleotides which hybridized to the sequence to be amplified depended on the melting temperature of the primers, which was determined as described by Breslauer et al. [PNAS USA (1986) 83:3746-50]. The average melting temperature of the selected oligos was 50-55° C. for the hybridizing region and 80-85° C. for the whole oligos.

C) Amplification

The standard PCR protocol was as follows: 50 ng genomic DNA were used as template in the presence of 0.5 μM each primer, 200 μM each dNTP, 1.5 mM MgCl₂, 1× buffer minus Mg⁺⁺ (Gibco-BRL) and 2 units of Taq DNA polymerase (Platinum Taq, Gibco-BRL) in a final volume of 100 μl. Each sample underwent a double-step of amplification: 5 cycles performed using as the hybridizing temperature 50° C., followed by 25 cycles at 68° C.

The standard cycles were as follows:

-   -   Denaturation: 94° C., 2 min     -   5 cycles: Denaturation: 94° C., 30 seconds     -   Hybridization: 50° C., 50 seconds     -   Elongation: 72° C., 1 min. or 2 min. and 40 sec.     -   25 cycles: Denaturation: 94° C., 30 seconds     -   Hybridization: 68° C., 50 seconds     -   Elongation: 72° C., 1 min. or 2 min. and 40 sec.

Elongation time was 1 minute for ORFs shorter than 2000 bp and 2:40 minutes for ORFs longer than 2000 bp. Amplifications were performed using a Gene Amp PCR system 9600 (Perkin Elmer).

To check amplification results, 2 μl of each PCR product were loaded onto 1-1.5 agarose gel and the size of amplified fragments was compared with DNA molecular weight standards (DNA marker IX Roche, 1 kb DNA ladder Biolabs).

Single band PCR products were purified by PEG precipitation: 300 μl of TE buffer and 200 μl of 30% PEG 8000/30 mM MgCl₂ were added to 100 μl PCR reaction. After vortexing, the DNA was centrifuged for 20 min at 10000 g, washed with 1 vol. 70% ethanol and the pellet dissolved in 30 μl TE. PCR products smaller than 350 bp were purified using a PCR purification Kit (Qiagen) and eluted with 30 μl of the provided elution buffer.

In order to evaluate the yield, 2 μl of the purified DNA were subjected to agarose gel electrophoresis and compared to titrated molecular weight standards.

D) Cloning of PCR Products into Expression Vectors

Cloning was performed following the GATEWAY™ technology's “one-tube protocol”, which consists of a two step reaction (BP and LR) for direct insertion of PCR products into expression vectors.

BP Reaction (attB×attP Sites):

The reaction allowed insertion of the PCR product into a pDONR vector. The pDONR™ 201 vector we used contains the killer toxin gene ccdB between attP1 and attP2 sites to minimize background colonies lacking the PCR insert, and a selectable marker gene for kanamycin resistance. The reaction resulted in a so called pEntry vector, in which the GBS gene was located between attL1 and attL2 sites.

60 fmol of PCR product and 100 ng of pDONR™ 201 vector were incubated with 2.5 μl of BP CLONASE™ in a final volume of 12.5 μl for 4 hours at 25° C.

LR Reaction (attL×attR Sites):

The reaction allowed the insertion of the GBS gene, now present in the pEntry vector, into E. coli expression vectors (pDestination vectors, containing attR sites). Two pDestination vectors were used (pDEST15 for N-terminal GST fusions—FIG. 86; and pDEST17-1 for N-terminal His-tagged fusions—FIG. 87). Both allow transcription of the ORF fusion coding mRNA under T7 RNA polymerase promoter [Studier et al (1990) Meth. Enzymol 185: 60ff].

To 5 μl of BP reaction were added 0.25 μl of 0.75 M NaCl, 100 ng of destination vector and 1.5 μl of LR CLONASE™. The reaction was incubated at 25° C. for 2 hours and stopped with 1 μl of 1 mg/ml proteinase K solution at 37° C. for 15 min.

1 μl of the completed reaction was used to transform 50 μl electrocompetent BL21-SI™ cells (0.1 cm, 200 ohms, 25 μF). BL21-SI cells contain an integrated T7 RNA polymerase gene under the control of the salt-inducible prU promoter [Gowrishankar (1985) J. Bacteriol. 164:434ff]. After electroporation cells were diluted in 1 ml SOC medium (20 g/l bacto-tryptone, 5 g/l yeast extract, 0.58 g/l NaCl, 0.186 g/l KCl, 20 mM glucose, 10 mM MgCl₂) and incubated at 37° C. for 1 hour. 200 μl cells were plated onto LBON plates (Luria Broth medium without NaCl) containing 100 μg/ml ampicillin. Plates were then incubated for 16 hours at 37° C.

Entry Clones:

In order to allow the future preparation of Gateway compatible pEntry plasmids containing genes which might turn out of interest after immunological assays, 2.5 μl of BP reaction were incubated for 15 min in the presence of 3 μl 0.15 mg/ml proteinase K solution and then kept at −20° C. The reaction was in this way available to transform E. coli competent cells so as to produce Entry clones for future introduction of the genes in other Destination vectors.

E) Protein Expression

Single colonies derived from the transformation of LR reactions were inoculated as small-scale cultures in 3 ml LBON 100 μg/ml ampicillin for overnight growth at 25° C. 50-200 μl of the culture was inoculated in 3 ml LBON/Amp to an initial OD600 of 0.1. The cultures were grown at 37° C. until OD600 0.4-0.6 and recombinant protein expression was induced by adding NaCl to a final concentration of 0.3 M. After 2 hour incubation the final OD was checked and the cultures were cooled on ice. 0.5 OD₆₀₀ of cells were harvested by centrifugation. The cell pellet was suspended in 50 μl of protein Loading Sample Buffer (50 mM TRIS-HCl pH 6.8, 0.5% w/v SDS, 2.5% v/v glycerin, 0.05% w/v Bromophenol Blue, 100 mM DTT) and incubated at 100° C. for 5 min. 10 μl of sample was analyzed by SDS-PAGE and Coomassie Blue staining to verify the presence of induced protein band.

F) Purification of the Recombinant Proteins

Single colonies were inoculated in 25 ml LBON 100 μg/ml ampicillin and grown at 25° C. overnight. The overnight culture was inoculated in 500 ml LBON/amp and grown under shaking at 25° C. until OD₆₀₀ values of 0.4-0.6. Protein expression was then induced by adding NaCl to a final concentration of 0.3 M. After 3 hours incubation at 25° C. the final OD₆₀₀ was checked and the cultures were cooled on ice. After centrifugation at 6000 rpm (JA10 rotor, Beckman) for 20 min., the cell pellet was processed for purification or frozen at −20° C.

Proteins were purified in 1 of 3 ways depending on the fusion partner and the protein's solubility:

Purification of Soluble His-Tagged Proteins from E. coli

1. Transfer pellets from −20° C. to ice bath and reconstitute each pellet with 10 ml B-PER™ solution (Bacterial-Protein Extraction Reagent, Pierce cat. 78266), 10 μl of a 100 mM MgCl₂ solution, 50 μl of DNAse I (Sigma D-4263, 100 Kunits in PBS) and 100 μl of 100 mg/ml lysozyme in PBS (Sigma L-7651, final concentration 1 mg/ml).

2. Transfer resuspended pellets in 50 ml centrifuge tubes and leave at room temperature for 30-40 minutes, vortexing 3-4 times.

3. Centrifuge 15-20 minutes at about 30-40000×g.

4. Prepare Poly-Prep (Bio-Rad) columns containing 1 ml of Fast Flow Ni-activated Chelating Sepharose (Pharmacia). Equilibrate with 50 mM phosphate buffer, 300 mM NaCl, pH 8.0.

5. Store the pellet at −20° C., and load the supernatant on to the columns.

6. Discard the flow through.

7. Wash with 10 ml 20 mM imidazole buffer, 50 mM phosphate, 300 mM NaCl, pH 8.0.

8. Elute the proteins bound to the columns with 4.5 ml (1.5 ml+1.5 ml+1.5 ml) 250 mM imidazole buffer, 50 mM phosphate, 300 mM NaCl, pH 8.0 and collect three fractions of ˜1.5 ml each. Add to each tube 15 μl DTT 200 mM (final concentration 2 mM).

9. Measure the protein concentration of the collected fractions with the Bradford method and analyse the proteins by SDS-PAGE.

10. Store the collected fractions at +4° C. while waiting for the results of the SDS-PAGE analysis.

11. For immunisation prepare 4-5 aliquots of 20-100 μg each in 0.5 ml in 40% glycerol. The dilution buffer is the above elution buffer, plus 2 mM DTT. Store the aliquots at −20° C. until immunisation.

Purification of His-Tagged Proteins from Inclusion Bodies

1. Bacteria are collected from 500 ml cultures by centrifugation. If required store bacterial pellets at −20° C. Transfer the pellets from −20° C. to room temperature and reconstitute each pellet with 10 ml B-PER™ solution, 10 μl of a 100 mM MgCl₂ solution (final 1 mM), 50 μl of DNAse I equivalent to 100 Kunits units in PBS and 100 μl of a 100 mg/ml lysozyme (Sigma L-7651) solution in PBS (equivalent to 10 mg, final concentration 1 mg/ml).

2. Transfer the resuspended pellets in 50 ml centrifuge tubes and let at room temperature for 30-40 minutes, vortexing 3-4 times.

3. Centrifuge 15 minutes at 30-4000×g and collect the pellets.

4. Dissolve the pellets with 50 mM TRIS-HCl, 1 mM TCEP {Tris(2-carboxyethyl)-phosphine hydrochloride, Pierce}, 6M guanidine hydrochloride, pH 8.5. Stir for ˜10 min. with a magnetic bar.

5. Centrifuge as described above, and collect the supernatant.

6. Prepare Poly-Prep (Bio-Rad) columns containing 1 ml of Fast Flow Ni-activated Chelating Sepharose (Pharmacia). Wash the columns twice with 5 ml of H₂O and equilibrate with 50 mM TRIS-HCl, 1 mM TCEP, 6M guanidine hydrochloride, pH 8.5.

7. Load the supernatants from step 5 onto the columns, and wash with 5 ml of 50 mM TRIS-HCl buffer, 1 mM TCEP, 6M urea, pH 8.5

8. Wash the columns with 10 ml of 20 mM imidazole, 50 mM TRIS-HCl, 6M urea, 1 mM TCEP, pH 8.5. Collect and set aside the first 5 ml for possible further controls.

9. Elute proteins bound to columns with 4.5 ml buffer containing 250 mM imidazole, 50 mM TRIS-HCl, 6M urea, 1 mM TCEP, pH 8.5. Add the elution buffer in three 1.5 ml aliquots, and collect the corresponding three fractions. Add to each fraction 15 μl DTT (final concentration 2 mM).

10. Measure eluted protein concentration with Bradford method and analyse proteins by SDS-PAGE.

11. Dialyse overnight the selected fraction against 50 mM Na phosphate buffer, pH 8.8, containing 10% glycerol, 0.5 M arginine, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, 2 M urea.

12. Dialyse against 50 mM Na phosphate buffer, pH 8.8, containing 10% glycerol, 0.5 M arginine, 5 mM reduced glutathione, 0.5 mM oxidized glutathione.

13. Clarify the dialysed protein preparation by centrifugation and discard the non-soluble material and measure the protein concentration with the Bradford method.

14. For each protein destined to the immunization prepare 4-5 aliquot of 20-100 μg each in 0.5 ml after having adjusted the glycerol content up to 40%. Store the prepared aliquots at −20° C. until immunization.

Purification of GST-Fusion Proteins from E. coli

1. Bacteria are collected from 500 ml cultures by centrifugation. If required store bacterial pellets at −20° C. Transfer the pellets from −20° C. to room temperature and reconstitute each pellet with 10 ml B-PER™ solution, 10 μl of a 100 mM MgCl₂ solution (final 1 mM), 50 μl of DNAse I equivalent to 100 Kunits units in PBS and 100 μl of a 100 mg/ml lysozyme (Sigma L-7651) solution in PBS (equivalent to 10 mg, final concentration 1 mg/ml).

2. Transfer the resuspended pellets in 50 ml centrifuge tubes and let at room temperature for 30-40 minutes, vortexing 3-4 times.

3. Centrifuge 15-20 minutes at about 30-40000×g.

4. Discard centrifugation pellets and load supernatants onto the chromatography columns, as follows.

5. Prepare Poly-Prep (Bio-Rad) columns containing 0.5 ml of Glutathione-Sepharose 4B resin. Wash the columns twice with 1 ml of H₂O and equilibrate with 10 ml PBS, pH 7.4.

6. Load supernatants on to the columns and discard the flow through.

7. Wash the columns with 10 ml PBS, pH 7.4.

8. Elute proteins bound to columns with 4.5 ml of 50 mM TRIS buffer, 10 mM reduced glutathione, pH 8.0, adding 1.5 ml+1.5 ml+1.5 ml and collecting the respective 3 fractions of ˜1.5 ml each.

9. Measure protein concentration of the fractions with the Bradford method and analyse the proteins by SDS-PAGE.

10. Store the collected fractions at +4° C. while waiting for the results of the SDS-PAGE analysis.

11. For each protein destined for immunisation prepare 4-5 aliquots of 20-100 μg each in 0.5 ml of 40% glycerol. The dilution buffer is 50 mM TRIS-HCl, 2 mM DTT, pH 8.0. Store the aliquots at −20° C. until immunisation.

FIG. 4

For the experiment shown in FIG. 4, the GBS proteins were fused at the N-terminus to thioredoxin and at C-terminus to a poly-His tail. The plasmid used for cloning is pBAD-DEST49 (Invitrogen Gateway™ technology) and expression is under the control of an L(+)-Arabinose dependent promoter. For the production of these GBS antigens, bacteria are grown on RM medium (6 g/l Na₂HPO₄, 3 g/l KH₂PO₄, 0.5 g/l NaCl, 1 g/l NH₄Cl, pH7.4, 2% casaminoacids, 0.2% glucose, 1 mM MgCl₂) containing 100 μg/ml ampicillin. After incubation at 37° C. until cells reach OD₆₀₀=0.5, protein expression is induced by adding 0.2% (v/v) L(+)Arabinose for 3 hours.

Immunisations with GBS Proteins

The purified proteins were used to immunise groups of four CD-1 mice intraperitoneally. 20 μg of each purified protein was injected in Freund's adjuvant at days 1, 21 & 35. Immune responses were monitored by using samples taken on day 0 & 49. Sera were analysed as pools of sera from each group of mice.

FACScan Bacteria Binding Assay Procedure.

GBS serotype V 2603 V/R strain was plated on TSA blood agar plates and incubated overnight at 37° C. Bacterial colonies were collected from the plates using a sterile dracon swab and inoculated into 100 ml Todd Hewitt Broth. Bacterial growth was monitored every 30 minutes by following OD₆₀₀. Bacteria were grown until OD₆₀₀=0.7-0.8. The culture was centrifuged for 20 minutes at 5000 rpm. The supernatant was discarded and bacteria were washed once with PBS, resuspended in ½ culture volume of PBS containing 0.05% paraformaldehyde, and incubated for 1 hour at 37° C. and then overnight at 4° C.

50 μl bacterial cells (OD₆₀₀ 0.1) were washed once with PBS and resuspended in 20 μl blocking serum (Newborn Calf Serum, Sigma) and incubated for 20 minutes at room temperature. The cells were then incubated with 100 μl diluted sera (1:200) in dilution buffer (20% Newborn Calf Serum 0.1% BSA in PBS) for 1 hour at 4° C. Cells were centrifuged at 5000 rpm, the supernatant aspirated and cells washed by adding 200 μl washing buffer (0.1% BSA in PBS). 50 μl R-Phicoerytrin conjugated F(ab)₂ goat anti-mouse, diluted 1:100 in dilution buffer, was added to each sample and incubated for 1 hour at 4° C. Cells were spun down by centrifugation at 5000 rpm and washed by adding 200 μl of washing buffer. The supernatant was aspirated and cells resuspended in 200 μl PBS. Samples were transferred to FACScan tubes and read. The condition for FACScan setting were: FL2 on; FSC-H threshold:54; FSC PMT Voltage: E 02; SSC PMT: 516; Amp. Gains 2.63; FL-2 PMT: 728. Compensation values: 0.

Samples were considered as positive if they had a Δ mean values >50 channel values.

Whole Extracts Preparation

GBS serotype III COH1 strain and serotype V 2603 V/R strain cells were grown overnight in Todd Hewitt Broth. 1 ml of the culture was inoculated into 100 ml Todd Hewitt Broth. Bacterial growth was monitored every 30 minutes by following OD₆₀₀. The bacteria were grown until the OD reached 0.7-0.8. The culture was centrifuged for 20 minutes at 5000 rpm. The supernatant was discarded and bacteria were washed once with PBS, resuspended in 2 ml 50 mM Tris-HCl, pH 6.8 adding 400 units of Mutanolysin (Sigma-Aldrich) and incubated 3 hrs at 37° C. After 3 cycles of freeze/thaw, cellular debris were removed by centrifugation at 14000 g for 15 minutes and the protein concentration of the supernatant was measured by the Bio-Rad Protein assay, using BSA as a standard.

Western Blotting

Purified proteins (50 ng) and total cell extracts (25 μg) derived from GBS serotype III COH1 strain and serotype V 2603 V/R strain were loaded on 12% or 15% SDS-PAGE and transferred to a nitrocellulose membrane. The transfer was performed for 1 hours at 100V at 4° C., in transferring buffer (25 mM Tris base, 192 mM glycine, 20% methanol). The membrane was saturated by overnight incubation at 4° C. in saturation buffer (5% skimmed milk, 0.1% TWEEN® 20 (polyoxyethylene sorbitan monolaurate) in PBS). The membrane was incubated for 1 hour at room temperature with 1:1000 mouse sera diluted in saturation buffer. The membrane was washed twice with washing buffer (3% skimmed milk, 0.1% TWEEN® 20 in PBS) and incubated for 1 hour with a 1:5000 dilution of horseradish peroxidase labelled anti-mouse Ig (Bio-Rad). The membrane was washed twice with 0.1% TWEEN® 20 in PBS and developed with the Opti-4CN Substrate Kit (Bio-Rad). The reaction was stopped by adding water.

Unless otherwise indicated, lanes 1, 2 and 3 of blots in the drawings are: (1) the purified protein; (2) GBS-III extracts; and (3) GBS-V extracts. Molecular weight markers are also shown.

In Vivo Passive Protection Assay in Neonatal Sepsis Mouse Model.

The immune sera collected from the CD1 immunized mice were tested in a mouse neonatal sepsis model to verify their protective efficacy in mice challenged with GBS serotype III. Newborn Balb/C littermates were randomly divided in two groups within 24 hrs from birth and injected subcutaneously with 25 μl of diluted sera (1:15) from immunized CD1 adult mice. One group received preimmune sera, the other received immune sera. Four hours later all pups were challenged with a 75% lethal dose of the GBS serotype III COH1 strain. The challenge dose obtained diluting a mid log phase culture was administered subcutaneously in 25 μl of saline. The number of pups surviving GBS infection was assessed every 12 hours for 4 days.

The contents of PCT publication WO2002/034771, including Examples 1 to 1374, and 1376 to 3329, all Figures, and Tables I to VI, is hereby incorporated by reference in its entirety.

Identification of GBSx 1460

A DNA sequence (GBSx1460) was identified in S. agalactiae <SEQ ID 4209> which encodes the amino acid sequence <SEQ ID 4210>. Analysis of this protein sequence reveals the following:

Possible site: 59 >>> Seems to have no N-terminal signal sequence ----- Final Results -----      bacterial cytoplasm --- Certainty = 0.1109 (Affirmative)      <succ>       bacterial membrane --- Certainty = 0.0000 (Not Clear)       <succ>        bacterial outside --- Certainty = 0.0000 (Not Clear)        <succ> The protein has homology with the following sequences in the GENPEPT database.

>GP:CAB73943 GB:AL139078 hyopthetical protein Cj1523c [Campylobacter jejuni] Identities = 165/746 (22%), Positives = 291/746 (38%), Gaps = 115/746 (15%) Query: 318 LSASMIQRYDEHREDLKQLKQFVKASLPEKYQEI--FADSSKDGYAGYIEGKINQEAFYK  375 L+ S  +R    +  L  LK  +       Y++   F +S    Y G +      E  ++ Sbjct:  50 LARSARKRLARRKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISP--YELRFR  107 Query: 376 YLSKLLIKQEDSENFLE--KIKNEDFLRKQRIFDNGSIPHQVHLIELKAIIRRQS-----  428  L++LL+KQ+ +   L   K +  D ++     + G+I   +   E K +   QS Sbjct: 108 ALNELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEK-LANYQSVGEYL  166 Query: 429 --EYYPFLKENQDRIEKILIFRIPYY-----------IGPLAREKSDFAW-MTRKIDDSI  474   EY+   KEN      +   +  Y            +  + +++ +F +  ++K ++ + Sbjct: 167 YKEYFQKFKENSKEFINVRNKKESYERCIAQSFLKDELKLIFKKQREFGFSFSKKFEEEV  226 Query: 475 RPWNFEDLVDKEKSAEAFIHRMINNDFYLPEEKVLPKHSLIYEKFIVYNELIKV--RYKN  532     F      +++ + F H + N  F+  +EK  PK+S +   F     +  +    KN  Sbjct: 227 LSVAFY-----KRALKDFSHLVGNCSFFI-DEKRAPKNSPLAFMFVALIRIINLLNNLKN  280 Query: 533 EQGEIYFFDSNIKQEIFDGVFKEHRKVSK--KKLLDFLAKEYEEFRIVDVIGLDKENKAF  590  +G  Y  D      + + V K      K  KKLL  L+ +YE            E   + Sbjct: 281 IEGILYIKDD--LNALLNEVLKNGILIYKQIKKLLG-LSDDYE---------FKGEKGIY  328 Query: 591 NASLGIYHDLEKILDKDFLDNPDNESILEDIVQTLTLFEDREMIKKRLENYKDLFIESQL  650       Y +  K L +  L   D    L +I + +TL +D   +KK L  Y     ++Q+ Sbjct: 329 FIEFKKYKEFIKALGEHNLSQDD----LNEIAKDIILIKDEIKLKKALAKYD--LNQNQI  382 Query: 651 KKLYRRHYIGWGRLSAKLINGIRDK--ESQKIILDYLIDDGRSNRNFMQLINDDGLSFKS  708   L +  +     +S K +  +     E +K       D+  +  N    IN+D   F Sbjct: 383 DSLSKLEFKDHLNISFKALKLVIPLMLEGKK------YDEACNELNLKVAINEDKKDFLP  436 Query: 709 IISKAQAGSHSDNLKEVVGELAGSPAIKKGILQSLKIVDELVKVMGYEPEQIVVEMAREN  768   ++        N           P + + I +  K+++ L+K  G +  +I +E+ARE Sbjct: 437 AFNEIYYKDEVIN-----------PVVLRAIKEYRKVLNALLKKYG-KVHKINIELAREV  484 Query: 769 QIINQGR----RNSRQRYKLLDDG---VKNLASDLNG-NILKEYPIDNQALQNERLFLYY  820    +  R    +   + YK   D     + L   +N  NILK             L L+ Sbjct: 485 GKNHSQRAKIEKEQNENYKAKKDAELECEKLGLKINSKNILK-------------LRLFK  531 Query: 821 LQNGRDMYIGEALDIDNLSQ---YDIDHIIPQAFIKDDSIDNRVLVSSAKNRGKSDDVPS  877  Q     Y+GE + I +L      +IDHI P +   DDS  N+VLV + +N+ K +  P Sbjct: 532 EQKEFCAYSGEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVLVFIKQNQEKLNQIP-  590 Query: 878 LEIVKDCKVFWKKL--LDAKLMSQRKYDNLTKAERGGLISDDKARFIQRQLVETRQIIKH  935  E   +    W+K+  L   L ++++   L K         ++  F  R L +TR I + Sbjct: 591 FEAFGNDSAKWQKIEVLAKNLPIKKQKRILDK----NYKDKEQKNFKDRNLNDIRYIARL  646 Query: 936 VARI---------LDERFNNELDSKGRRIRKVKIVTLKSNLVSNFRKEFGFYKIREVNNY  986 V            L +  N +L+   ++  KV +      L S  R  +GF      N+ Sbjct: 647 VLNYIKDYLDFLPLSDDENIKLNDI-QKGSKVHVEAKSGMLISALRHIWGFSAKDRNNHL  705 Query: 987 HHAHDAYLNAVVAKAILIKYPQLEPE 1012 HHA DA + A    +I+  +  +  E Sbjct: 706 HHAIDAVIIAYANNSIVKAFSDFKKE  731

A related DNA sequence was identified in S. pyogenes <SEQ ID 4211> which encodes the amino acid sequence <SEQ ID 4212>. Analysis of this protein sequence reveals the following:

Possible site: 61 >>> Seems to have no N-terminal signal sequence ----- Final Results -----      bacterial cytoplasm --- Certainty = 0.0973 (Affirmative)      <succ>       bacterial membrane --- Certainty = 0.0000 (Not Clear)       <succ>        bacterial outside --- Certainty = 0.0000 (Not Clear)        <succ>

An alignment of the GAS and GBS proteins is shown below.

Identities = 881/1380 (63%), Positives = 1088/1380 (78%), Gaps = 22/1380 (1%) Query: 1 MNKPYSIGLDIGTNSVGWSIITDDYKVPAKKMRVLGNTDKEYIKKNLIGALLFDGGNTAA   60 M+K YSIGLDIGTNSVGW++ITD+YKVP+KK +VLGNTD+  IKKNLIGALLFD G TA Sbjct: 1 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE   60 Query: 61 DRRLKRTARRRYTRRRNRILYLQEIFAEEMSKVDDSFFHRLEDSFLVEEDKRGSKYPIFA  120   RLKRTARRRYTRR+NRI YLQEIF+ EM+KVDDSFFHRLE+SFLVEEDK+  ++PIF Sbjct: 61 AIRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG  120 Query: 121 ILQEEKDYHEKFSTIYHLRKELADKKEKADLRLIYIALAHIIKFRGHFLIEDDSFDVRNT  180  + +E  YHEK+ TIYHLRK+L D  +KADLRLIY+ALAH+IKFRGHFLIE D  +  N+ Sbjct: 121 NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGD-LNPDNS  179 Query: 181 DISKQYQDFLEIFNTTFENNDLLSQNVDVEAILTDKISKSAKKDRILAQYPNQKSTGIFA  240 D+ K +   ++ +N  FE N + +  VD +AIL+ ++SKS + + ++AQ P +K G+F Sbjct: 180 DVDKLFIQLVQIYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFG  239 Query: 241 EFLKLIVGNQADFKKYFNLEDKTPLQFAKDSYDEDLENLLGQIGDEFADLFSAAKKLYDS  300   + L +G   +FK  F+L +   LQ +KD+YD+DL-NLL QIGD++ADLF AAK L D+ Sbjct: 240 NLIALSLGLIPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDA  299 Query: 301 VLLSGILIVIDLSTKAPLSASMIQRYDEHREDLKQLKQFVKASLPEKYQEIFADSSKDGY  360 +LLS IL V    TKAPLSASMI+RYDEH +DL  LK  V+  LPEKY+EIF D SK+GY Sbjct: 300 ILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGY  359 Query: 361 AGYIEGKTNQEAFYKYLSKLLTKQEDSENFLEKIKNEDFLRKQRTFDNGSIPHQVHLTEL  420 AGYI+G  +QE FYK++  +L K + +E  L K+  ED LRKQRTFDNGSIPHQ+HL EL Sbjct: 360 AGYIDGGASQEEFYKFIKPILEKMDGIEELLVKLNREDLLRKQRTFDNGSIPHQIHLGEL  419 Query: 421 KAIIRRQSEYYPFLKENQDRIEKILTFRIPYYIGPLAREKSDFAWMIRKIDDSIRPWNFE  480  AI+RRQ ++YPFLK+N+++IEKILTFRIPYY+GPLAR  S FAWMIRK++++I PWNFE Sbjct: 420 HAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMIRKSEETITPWNFE  479 Query: 481 DLVDKEKSAEAFIHRMTNNDFYLPEEKVLPKHSLIYEKFTVYNELTKVRYKNE-QGETYF  539 ++VDK  SA++FI RMTN D  LP EKVLPKHSL+YE FTVYNELTKV+Y  E  +   F Sbjct: 480 EVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAF  539 Query: 540 FDSNIKQEIFDGVFKEHRKVSKKKLLDFLAKEYEEFRIVDVIGLDKENKAFNASLGTYHD  599      K+ I D +FK +RKV+ K+L +   K+ E F  V++ G++     FNASLGTYHD SbJct: 540 LSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDR---FNASLGTYHD  596 Query: 600 LEKIL-DKDFLDNPDNESILEDIVQTLTLFEDREMIKKRLENYKDLFTESQLKKLYRRHY  658 L KI+ DKDFLDN +NE ILEDIV TLTLFEDREMI++RL+ Y  LF +  +K+L RR Y Sbjct: 597 LLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY  656 Query: 659 TGWGRLSAKLINGIRDKESQKTILDYLIDDGRSNRNFMQLINDDGLSFKSIISKAQAGSH  718 TGWGRLS KLINGIRDK+S KTILD+L  DG +NRNFMQLI+DD L+FK  I KAQ Sbjct: 657 TGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQ  716 Query: 719 SDNLKEVVGELAGSPAIKKGILQSLKIVDELVKVMG-YEPEQIVVEMARENQTTNQGRRN  777  D+L E +  LAGSPAIKKGILQ++K+VDELVKVMG ++PE IV+EMARENQTT +G++N Sbjct: 717 GDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKN  776 Query: 778 SRQRYKLLDDGVKNLASDLNGNILKEYPIDNQALQNERLFLYYLQNGRDMYIGEALDIDN  837 SR+R K +++G+K L S     ILKE+P +N  LQNE+L+LYYLQNGRDMY  + LDI+ Sbjct: 777 SRERMKRIEEGIKELGS----QILKEHPVENIQLQNEKLYLYYLQNGRDMYVDQELDINR  832 Query: 838 LSQYDIDHIIPQAFIKDDSIDNRVLVSSAKNRGKSDDVPSLEIVKDCKVFWKKLLDAKLM  897 LS YD+DHI+PQ+F+KDDSIDN+VL  S KNRGKSD+VPS E+VK  K +W++LL+AKL+ Sbjct: 833 LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLI  892 Query: 898 SQRKYDNLTKAERGGLTSDDKARFIQRQLVEIRQITKHVARILDERFNNELDSKGRRIRK  957 +QRK+DNLTKAERGGL+  DKA FI+RQLVEIRQITKHVA+ILD R N + D   + IR+ Sbjct: 893 TQRKFDNLTKAERGGLSELDKAGFIKRQLVEIRQITKHVAQILDSRMNIKYDENDKLIRE  952 Query: 958 VKIVTLKSNLVSNFRKEFGFYKIREVNNYHHAHDAYLNAVVAKAILTKYPQLEPEFVYGD 1017 VK++TLKS LVS+FRK+F FYK+RE+NNYHHAHDAYLNAVV  A++ KYP+LE EFVYGD Sbjct: 953 VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGIALTKKYPKLESEFVYGD 1012 Query: 1018 YPKYN-------SYKIRKSATEKLFFYSNIMNFFKTKVTLADGTVVVKDDIEVNNDTGEI 1070 Y  Y+       S +    AT K FFYSNIMNFFKT++TLA+G +  +  IE N +IGEI Sbjct: 1013 YKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI 1072 Query: 1071 VWDKKKHFATVRKVLSYPQNNIVKKTEIQTGGFSKESILAHGNSDKLIPRKTKDIYLDPK 1130 VWDK + FATVRKVLS PQ NIVKKTE+QTGGFSKESIL   NSDKLI RK KD   DPK Sbjct: 1073 VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARK-KD--WDPK 1129 Query: 1131 KYGGFDSPIVAYSVLVVADIKKGKAQKLKIVIELLGITIMERSRFEKNPSAFLESKGYLN 1190 KYGGFDSP VAYSVLVVA ++KGK++KLK+V ELLGITIMERS FEKNP  FLE+KGY Sbjct: 1130 KYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE 1189 Query: 1191 IRADKLIILPKYSLFELENGRRRLLASAGELQKGNELALPTQFMKFLYLASRYNESKGKP 1250 ++ D +I LPKYSLFELENGR+R+LASAGELQKGNELALP++++ FLYLAS Y + KG P Sbjct: 1190 VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSP 1249 Query: 1251 EEIEKKQEFVNQHVSYFDDILQLINDFSKRVILADANLEKINKLYQDNKENISVDELANN 1310 E+ E+KQ FV QH Y D+I++ I++FSKRVILADANL+K+    Y  +++   + E A N Sbjct: 1250 EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK-PIREQAEN 1308 Query: 1311 IINLFTFTSLGAPAAFKFFDKIVDRKRYTSTKEVLNSTLIHQSITGLYETRIDLGKLGED 1370 II+LFT T+LGAPAAFK+FD  +DRKRYTSTKEVL++TLIHQSITGLYETRIDL +LG D Sbjct: 1309 IIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 1368

SEQ ID 4210 (GBS317) was expressed in E. coli as a GST-fusion product. SDS-PAGE analysis of total cell extract is shown in FIG. 27 (lane 2; MW 179.3 kDa) and in FIG. 159 (lane 5 & 6; MW 180 kDa). It was also expressed in E. coli as a His-fusion product. SDS-PAGE analysis of total cell extract is shown in FIG. 27 (lane 3; MW 154.3 kDa) and in FIG. 159 (lane 9 & 10; MW 154 kDa).

GBS317-GST was purified as shown in FIG. 224, lane 9-10. GBS317-His was purified as shown in FIG. 222, lane 9.

GBS317N was expressed in E. coli as a GST-fusion product. SDS-PAGE analysis of total cell extract is shown in FIG. 149 (lane 2-4; MW 116 kDa).

GBS317C was expressed in E. coli as a GST-fusion product. SDS-PAGE analysis of total cell extract is shown in FIG. 166 (lane 6-8; MW 92 kDa).

GBS317dN was expressed in E. coli as a GST-fusion product. SDS-PAGE analysis of total cell extract is shown in FIG. 187 (lane 7; MW 116 kDa). Purified GBS317dN-GST is shown in FIG. 245, lane 8.

GBS317C was expressed in E. coli as a GST-fusion product. SDS-PAGE analysis of total cell extract is shown in FIG. 188 (lane 13; MW 92 kDa). Purified GBS317dC-GST is shown in FIG. 245, lane 9.

Based on this analysis, it was predicted that these proteins and their epitopes could be useful antigens for vaccines or diagnostics.

TABLE I THEROETICAL MOLECULAR WEIGHTS FOR GBS PROTEINS expected mol. weight (dalton) GBS # GST-fusion His-fusion Native  1 78425 53460 49720  2 40035 15070 11330  3 90305 65340 61600  4 43115 18150 14410  5 158835 133870 130130  6 39265 14300 10560  7 44985 20020 16280  8 56315 31350 27610  9 50265 25300 21560  10 96465 71500 67760  11 91515 66550 62810  11d 85905 60940 57200  12 64455 39490 35750  13 40475 15510 11770  14 33325 8360 4620  15 44765 19800 16060  16 73475 48510 44770  17 46745 21780 18040  18 54335 29370 25630  19 46085 21120 17380  20 47625 22660 18920  21 56535 31570 27830  21 long 66435 41470 37730  22 60055 35090 31350  23 60165 35200 31460  24 58405 33440 29700  25 50265 25300 21560  26 118245 93280 89540  28 63795 38830 35090  29 50595 25630 21890  30 44215 19250 15510  31 63795 38830 35090  31d 58735 33770 30030  32 40585 15620 11880  33 71495 46530 42790  34 69295 44330 40590  35 56535 31570 27830  36 59065 34100 30360  37 46965 22000 18260  38 61815 36850 33110  39 65225 40260 36520  41 75235 50270 46530  42 46745 21780 18040  43 58955 33990 30250  44 52355 27390 23650  45 43555 18590 14850  46 59835 34870 31130  47 84255 59290 55550  48 86455 61490 57750  48d 106695 81730 77990  49 59615 34650 30910  50 94155 69190 65450  51 47075 22110 18370  52 55435 30470 26730  53 110215 85250 81510  54 73365 48400 44660  55 36295 11330 7590  56 34865 9900 6160  57 51145 26180 22440  58 128805 103840 100100  59 99215 74250 70510  60 63575 38610 34870  61 68085 43120 39380  62 105485 80520 76780  63 64125 39160 35420  64 112745 87780 84040  65 72485 47520 43780  66 49715 24750 21010  67 120335 95370 91630  68 131225 106260 102520  68d 103065 78100 74360  69 53895 28930 25190  70 74465 49500 45760  70d 59725 34760 31020  71 56755 31790 28050  72 75565 50600 46860  73 72815 47850 44110  74 131225 106260 102520  74d 95475 70510 66770  75 114725 89760 86020  76 198875 173910 170170  77 78535 53570 49830  78 48835 23870 20130  79 58185 33220 29480  79d 50815 25850 22110  80 81835 56870 53130  81 89205 64240 60500  82 40475 15510 11770  83 62585 37620 33880  84 122645 97680 93940  85 70175 45210 41470  86 84035 59070 55330  87 44435 19470 15730  88 73365 48400 44660  89 143325 118360 114620  90 93495 68530 64790  91 88325 63360 59620  92 193595 168630 164890  93 95585 70620 66880  94 77435 52470 48730  95 60605 35640 31900  96 57195 32230 28490  97 138375 113410 109670  98 82055 57090 53350  99 60715 35750 32010 100 53015 28050 24310 101 59395 34430 30690 102 40695 15730 11990 103 56975 32010 28270 104 120005 95040 91300 105 179735 154770 151030 105dNterm 127265 102300 98560 105dCterm 81285 56320 52580 106 85795 60830 57090 107 89535 64570 60830 108 64565 39600 35860 109 75125 50160 46420 109d 70725 45760 42020 110 53895 28930 25190 111/190 60165 35200 31460 112 63905 38940 35200 113 59175 34210 30470 114 51915 26950 23210 115 98225 73260 69520 116 73475 48510 44770 117 47515 22550 18810 118 42235 17270 13530 119 109225 84260 80520 120 71385 46420 42680 121 65115 40150 36410 122 46855 21890 18150 123 68305 43340 39600 124 54115 29150 25410 125 57305 32340 28600 126 56865 31900 28160 127 80845 55880 52140 128 39925 14960 11220 129 43775 18810 15070 130 82275 57310 53570 130d 63245 38280 34540 131 89755 64790 61050 132 49055 24090 20350 133 54445 29480 25740 134 42015 17050 13310 135 65225 40260 36520 136 54885 29920 26180 137 63465 38500 34760 138 40145 15180 11440 139 38165 13200 9460 140 43445 18480 14740 141 49935 24970 21230 142 79745 54780 51040 143 33545 8580 4840 144 49165 24200 20460 145 63025 38060 34320 146 107025 82060 78320 147 156965 132000 128260 148 41905 16940 13200 149 62365 37400 33660 150 54665 29700 25960 151 50412 25447 21707 151L 50045 25080 21340 152 45535 20570 16830 153 46965 22000 18260 154 101525 76560 72820 155 62585 37620 33880 156 61265 36300 32560 157 74025 49060 45320 158 52025 27060 23320 159 41025 16060 12320 160 82825 57860 54120 161 95365 70400 66660 162 42015 17050 13310 163 69405 44440 40700 164 42345 17380 13640 165 43555 18590 14850 166 38055 13090 9350 167 50375 25410 21670 168 32555 7590 3850 169 43445 18480 14740 170 64015 39050 35310 170d 59945 34980 31240 171 49825 24860 21120 172 62365 37400 33660 173 96795 71830 68090 174 45095 20130 16390 175 59175 34210 30470 176 55435 30470 26730 177 66215 41250 37510 178 62365 37400 33660 179 58515 33550 29810 180 37615 12650 8910 181 63685 38720 34980 182 90085 65120 61380 182d 87225 62260 58520 183 57855 32890 29150 184 46415 21450 17710 185 40695 15730 11990 186 85685 60720 56980 187 56205 31240 27500 188 61595 36630 32890 189 60165 35200 31460 191 116705 91740 88000 192 69625 44660 40920 193 98005 73040 69300 194 49385 24420 20680 195 81065 56100 52360 195L 147615 122650 118910 195L N-term 91405 66440 62700 196 69515 44550 40810 197 99325 74360 70620 198 73805 48840 45100 199 158285 133320 129580 200 132325 107360 103620 201 74538 49573 45833 202 157295 132330 128590 203 61705 36740 33000 204 39705 14740 11000 205 55985 31020 27280 206 56645 31680 27940 207 44765 19800 16060 208 59725 34760 31020 209 62145 37180 33440 209d 56425 31460 27720 210 60935 35970 32230 210d 53675 28710 24970 211 64895 39930 36190 212 60825 35860 32120 213 45205 20240 16500 214 38935 13970 10230 215 45205 20240 16500 216 91515 66550 62810 217 36075 11110 7370 218 81065 56100 52360 219 56535 31570 27830 220 54555 29590 25850 220 50155 25190 21450 221 41465 16500 12760 222 47405 22440 18700 223 42895 17930 14190 224 45865 20900 17160 225 56645 31680 27940 226 44875 19910 16170 227 46195 21230 17490 228 46525 21560 17820 229 35855 10890 7150 230 51915 26950 23210 231 60935 35970 32230 231d 58735 33770 30030 232 41795 16830 13090 233 35635 10670 6930 234 43115 18150 14410 235 58295 33330 29590 235d 48395 23430 19690 236 46525 21560 17820 237 44215 19250 15510 238 59725 34760 31020 239 63905 38940 35200 240 51475 26510 22770 241 45095 20130 16390 242 43225 18260 14520 243 119455 94490 90750 244 48065 23100 19360 245 48615 23650 19910 246 49605 24640 20900 246d 45975 21010 17270 247 58955 33990 30250 248 92505 67540 63800 248d 70835 45870 42130 249 103835 78870 75130 250 136505 111540 107800 251 52135 27170 23430 252 51695 26730 22990 253 74245 49280 45540 254 59615 34650 30910 255 69075 44110 40370 256 47845 22880 19140 257 60495 35530 31790 258 67975 43010 39270 259 79415 54450 50710 260 48175 23210 19470 261 55765 30800 27060 262 75345 50380 46640 263 63465 38500 34760 264 47185 22220 18480 265 56315 31350 27610 266 51365 26400 22660 267 88655 63690 59950 268 50265 25300 21560 269 60495 35530 31790 270 59285 34320 30580 271 56315 31350 27610 272 118355 93390 89650 272d 98885 73920 70180 273 70945 45980 42240 274 56205 31240 27500 275 47515 22550 18810 276 147945 122980 119240 277 87005 62040 58300 277d 75675 50710 46970 278 52245 27280 23540 279 79415 54450 50710 280 88655 63690 59950 281 74465 49500 45760 281d 71495 46530 42790 282 44765 19800 16060 283 20240 16500 284 67645 42680 38940 285 57525 32560 28820 286 41355 16390 12650 287 61045 36080 32340 287d 57085 32120 28380 288 53675 28710 24970 288d 51035 26070 22330 289 65005 40040 36300 289 long 71825 46860 43120 290 47405 22440 18700 291 63795 38830 35090 292 103505 78540 74800 293 115935 90970 87230 293d N-term 73805 48840 45100 293d C-term 70835 45870 42130 294 75785 50820 47080 295 89425 64460 60720 296 60385 35420 31680 297 100205 75240 71500 298 54335 29370 25630 299 62255 37290 33550 300 130895 105930 102190 301 54885 29920 26180 302 80075 55110 51370 303 53235 28270 24530 304 75125 50160 46420 305 78645 53680 49940 306 67975 43010 39270 307 86675 61710 57970 308 59285 34320 30580 309 62695 37730 33990 310 58845 33880 30140 311 76445 51480 47740 312 64785 39820 36080 313 65995 41030 37290 314 52135 27170 23430 315 51695 26730 22990 316 41795 16830 13090 317 179295 154330 150590 317d N-term 115935 90970 87230 317d C-term 92160 67402 63360 318 70065 45100 41360 319 61925 36960 33220 320 57965 33000 29260 321 83705 58740 55000 322 76628 51663 47923 323 86345 61380 57640 324 86345 61380 57640 325 82605 57640 53900 326 91515 66550 62810 326L 172695 147730 143990 326L N-term 113955 88990 85250 327 279175 254210 250470 327d N-term 139915 114950 111210 327d C-term 167965 143000 139260 328 97602 72637 68897 329 113955 88990 85250 330 83595 58630 54890 331 60825 35860 32120 332 75675 50710 46970 333 63465 38500 34760 333d 57965 33000 29260 334 38275 13310 9570 335 43555 18590 14850 336 67645 42680 38940 337 75235 50270 46530 338 54995 30030 26290 339 76665 51700 47960 339d 72925 47960 44220 340 86565 61600 57860 341 38385 13420 9680 342 61595 36630 32890 343 60385 35420 31680 344 55875 30910 27170 345 40585 15620 11880 346 53895 28930 25190 347 55325 30360 26620 348 58405 33440 29700 349 98335 73370 69630 350 53895 28930 25190 351 82165 57200 53460 352 111315 86350 82610 352d 105485 80520 76780 353 55325 30360 26620 354 42345 17380 13640 355 52135 27170 23430 356 59065 34100 30360 357 40255 15290 11550 358 60495 35530 31790 359 78865 53900 50160 360 73695 48730 44990 361 109005 84040 80300 362 125945 100980 97240 362d N-term 63355 38390 34650 362d C-term 91295 66330 62590 363 53125 28160 24420 364 75015 50050 46310 365 102075 77110 73370 366 68415 43450 39710 367 76885 51920 48180 368 44765 19800 16060 369 142115 117150 113410 370 94595 69630 65890 371 65555 40590 36850 372 55105 30140 26400 373 50265 25300 21560 374 57525 32560 28820 375 66875 41910 38170 376 48065 23100 19360 377 73805 48840 45100 378 58955 33990 30250 379 68855 43890 40150 380 47405 22440 18700 381 66875 41910 38170 382 50815 25850 22110 383 57085 32120 28380 384 77985 53020 49280 385 75675 50710 46970 386 39485 14520 10780 387 54555 29590 25850 388 45645 20680 16940 389 43005 18040 14300 390 62255 37290 33550 391 54775 29810 26070 392 71385 46420 42680 393 55765 30800 27060 394 59725 34760 31020 395 72375 47410 43670 396 34865 9900 6160 397 113625 88660 84920 397d 100865 3740 72160 398 56755 31790 28050 399 55435 30470 26730 400 74135 49170 45430 401 59395 34430 30690 402 78095 53130 49390 403 64455 39490 35750 404 61595 36630 32890 405 45975 21010 17270 406 36955 11990 8250 407 82715 57750 54010 407d 71715 46750 43010 408 45315 20350 16610 409 70395 45430 41690 409d 59600 34842 30800 410 62475 37510 33770 411 41355 16390 12650 412 35965 11000 7260 413 59175 34210 30470 414 50375 25410 21670 415 46195 21230 17490 416 42455 17490 13750 417 77985 53020 49280 418 42125 17160 13420 419 47515 22550 18810 420 67755 42790 39050 421 62915 37950 34210 422 60165 35200 31460 423 74245 49280 45540 424 89975 65010 61270 424 77325 52360 48620 425 116045 91080 87340 426 83815 58850 55110 427 41135 16170 12430 428 55325 30360 26620 429 59175 34210 30470 430 53785 28820 25080 431 54005 29040 25300 432 65665 40700 36960 433 40915 15950 12210 434 44545 19580 15840 642 91845 66880 63140 643 78975 54010 50270 644 49605 24640 20900 645 59725 34760 31020 646 61595 36630 32890 647 55875 30910 27170 648 59835 34870 31130 649 76115 51150 47410 650 51475 26510 22770 651 53345 28380 24640 652 49715 24750 21010 653 44655 19690 15950 654 51255 26290 22550 655 65995 41030 37290 656 57525 32560 28820 657 62805 37840 34100 658 60165 35200 31460 659 60275 35310 31570 660 71495 46530 42790 661 60605 35640 31900 662 62695 37730 33990 663 89535 64570 60830 664 45315 20350 16610 665 41135 16170 12430 666 47075 22110 18370 667 53162 28197 24457 668 43555 18590 14850 669 48505 23540 19800 670 45315 20350 16610 671 36940 12182 8140 672 40130 15372 11330 673 41450 16692 12650 674 45300 20542 16500 675 55970 31212 27170 676 65650 40892 36850 677 54320 29562 25520 678 77750 52992 48950 679 60480 35722 31680 680 64440 39682 35640 681 93040 68282 64240 682 84790 60032 55990 683 15950 44655 19690 684 11880 40585 15620 685 16280 44985 20020 686 21340 50045 25080 687 9350 38055 13090 689 55105 3740 26400

TABLE II PRIMERS USED TO AMPLIFY GBSnnn PROTEINS Forward primers begin 5′-GGGGACAAGTTTGTACAAAAAAGCAGGC-3′ and continue with the sequences indicated in the table below; reverse primers begin  5′-GGGGACCACTTTGTACAAGAAAGCTGGGTT-3′ and continue with the sequences indicated in the table. The primers for GBS1 are thus: Fwd: GGGGACAAGTTTGTACAAAAAAGCAGGCTCTCAATCTCATATTGTTTCAG Rev: GGGGACCACTTTGTACAAGAAAGCTGGGTTATTTTTAGACATCATAGACA The full forward primer sequences are given in the sequence listing as SEQ IDs 10968-11492. The reverse primer sequences are SEQ IDs 11493-12017. GBS Forward Reverse 1 TCTCAATCTCATATTGTTTCAG ATTTTTAGACATCATAGACA 2 TCTAATTACATTATTACATTTT GGGAATGCCTACAAA TG 3 TCTGATACTAGTTCAGGAATATC TTTTTTACTATACTTTTTGT 4 TCTGATACAAGTGATAAGAATAC TTCCTTTTTAGGCTTACT T 5 TCTATTTTTCTTCATAGTCCAC ATTAGCTTCATTTGTCAG 6 TCTGAATGGGTGTTATTAACTC AGTTTCTTCTTTAAAATCAT 7 TCTACAAATTCTTATTTTAGCAA CTCTGAAGCTGTAAAACC 8 TCTGTATCAGTTCAGGCGT TTTATCAATGTTTGAAACG 9 TCTGCTGCTCTAGGACAAC TAGTAAATCAAGTTTTTGCA 10 TCTTTTGTTGTTGCCTTATT ATCCCTTCTATTTTCGA 11 TCTCCACCTATGGAACGT ATGTAGTGACGTTTCTGTG 11d TCTCAGAAAGTCTATCGGG ATGTAGTGACGTTTCTGTG 12 TCTAGTGAGAAGAAAGCAAAT ATTGGGTGTAAGCATT 13 TCTTCTTGGAATTATTGGAG CTTAACTCTACCCGTCC 14 TCTGCAATGATTGTAACCAT TTTTCTCTTATTAAAGAATT 15 TCTGCATCTTATACCGTGAA ATACCAGCCGTTACTATT 16 TCTGCCGAGAAGGATAAA TTTAGCTGCTTTTTTAATG 17 TCTGTTTATAAAGTTATTCAAAA AAATACTACATTTACAGGTG 18 TCTAAGCCTAACAGTCAACA TTGGTTATTCTCCTTTAAT 19 TCTGATGATAACTTTGAAATGC ATTATATTTTTGGATATTTC 20 TCTGCAGTGATTGCAAGTC GGGCTTTTTCTTAAAAA 21 TGTGCTGCATCAAAC GTTGGCATCCCTTTT 21  TGTGCTGCATCAAAC CTTTTGATGGGATTGG Long + A527 22 TGTACTAAACAAAGCCAG TTGATTTAACGATTTGA 23 TGTCAATTAACCGATAC TTTATCTCCTCTAAAATAATG 24 TGCTCAAATGATTCAT CTTTGATAAGTCAGACCA 25 TCTAAAAGTTCACAAGTTACTA GTAACCCCAAGCTGAT CT 26 TCTAGTCATTATTCCATAAAATT TGATTTTGCAATATCAA 28 TCTAATCATATGCTGATTGAG TTTTTGTAATTTAAGTACTAA 29 TCAGTTTGGATGTTAAC TTCTTTTATATTAAGAGCTT 30 TCAACAAATGCAGATG ATTCGGATAAATGTAGC 31 TGTTTTGTCATTATTGATAG TCCATTTTTATCCTCAC 31d TCTCTAACTTGGTTTTTATTAGA TCCATTTTTATCCTCAC 32 TCTGGTTTAAAAGTGACTGAA ATGACCTCTACTTTCCA 33 TCTCATCATTTAGGTAAGGAA CTTGTAATCACTTGGAC 34 TCTGTTAGTAATCGCTACAATC ATTAATCATGGTATTGGT 35 TCTAATCAAGAAGTTTCAGC CCATTGTGGAATATCA 36 TCTCGAGTTTTAGCGGATA TTTGTAAAGCAGTTCTT 37 TCTGTATTATTTTACCAATCACA ATCATTCATATGATCTCTAGA 38 TTAGGAGTGGTAGTTCAT ATTTTGATTGATTCTACTC 39 TTTTTATTGTTAGTATTAGC TTTTGTTTTTTTCAAATA 41 TCTGTTTATCTAGCGGTTAGA ATCTTCAACGTCCTCC 42 TATAACAGTTTAGTTAGAAGTC AAAGTCAAAGGAAACTT 43 TTTAAAGGGTTTACATATT TTCTTTATCTAATTTATAATA G 44 TTTAATACAATTGGTCG TTGCAATGTTTTTTCT 45 TCTATGGAAAAAATTAGGATT TAAACTTTGGATAATCTGT 46 TCTAGAGATGAGCAAGAAATA GTTGAAATTTTGATATGA 47 TCTCAACAGATAGGTCTTTATAA CTCCTTTACTATATAGCTAAC T 48 TTTCTCTATAATTACTTCAAT TTGTTTGTGAAGTAAAAC 49 TCTAATAAGGCATTATTAGAGG TGATAATATCTCCATATTTT 50 TCTACACATTTAGTTGACTTAAC GCATTGGCGCCATA 51 TCTAGTAAACAACACATTTATCT TTCTACACGACTTTTATTC A 52 TCTCAAGAAACTCATCAGTTG AAGACCTCCTCGAGAT 53 TCTGCAGAAGACATTGTTACA TGTTTTTTCTTTCTGTTG 54 TATAATTTTTCGACTAATGA TGGATTAGTTTGACCTG 55 TCTGACACAGTGTCTTATCCT TTTATCGTAAGCACTTAGG 56 TCTGTGGAGCAAGTGGCCA CTCCTTCCAGGCATCG 57 TCTCAAGAACTAAGTAACTTTGA GTAAAAGTATCTTAAATAGTC A 58 TCTACTGAAACGTTTGAAGG TGCCATTCCTCCTCT 59 TCTGATGAAGCAACAACTAA TGTTACCTTTTTATTTTCT 60 TCTAATAAAGATAATCAAAAAAC TTTTTCATGCGATTGA T 61 TGTTTCTTTTTTATTCCA GAGACGTTTCTTATACCTT 62 TATTACTTTGATGGTAGTTT TGTACCATATGTTCTCTCT 63 TCTGTTCAATCATTAGCAAA AAAAGTTGGACTACTTTC 64 TTTAAAGGTAATAAGAAGTTG TCGTTTTCCACCC 64d TCTAGTCAAGTTGACTCTGTTA TCGTTTTCCACCC 65 TCTCAAAACCAGGTGACTG ATTTGGGTAAATATAGTAAA 66 TTAAGATTTTATAACAACGA TTTACGACTAACCTCAAC 67 TCTAATGTTTTAGGGGAAA AATTCCTTTTGGTGG 68 TCCCAAAAGACTTTTG GGCAGAATACACCTTC 68d TCCCAAAAGACTTTTG GGCTGACGTCGACGCA 69 TCTAAAGTTTTAGCCTTTGA AACTCTCTTAATATATTCTTC T 70 TCTGAAATGGCTTTAG GTCTTTTTCAATATTCTGT 70d TCTACTAACTTATTGAGTAGAAT GTCTTTTTCAATATTCTGT CA 71 TGTAGCTCAAAATCTCAT CTTCTCCTTAGGAGTAACG 72 TCTAGTTTATCTATTAAAGATGC ATTATTATCAATTAATAACTC C TT 73 TCTATCAAAGAGGCGGTAA GTCAAACATACTTCCAAA 74 TCTAAAGAGGATAAAAAGCTAG TTTCGTCGTATAAGCA 74d TCTAGTGTTTCAGGTAGTAGTG TTTCGTCGTATAAGCA 75 TCTAAAAAATTAAAACACTCAA TGTCCTCATTTTTTCAG 76 TCTGATGAAGTTACAACTTCAG AATACTTGCTGGAACAG 77 TTATTCCAAAGTAAAATAAA GTCTTTCTTCAATTTTGG 78 TCTCATAACCATCACTCAGAAC GTCGTGATTTTTATGAGT ACATGT 79 TCTCCCAAGAATAGGATAAA CCCAAACTGGCATAAC 79d TCTAGTCAGTATGAGTCACAGA CCCAAACTGGCATAAC 80 TCTGCAGAAGTGTCACAAGA TGAAGGACGTTTGTTG 81 TCTTTTGATGGATTTTT TTTTTTTAGTTTAAGGCTA 82 TCTACAAATGAAAAACGAAC GTCCACCTTCCGAT 83 TCTGAAATTAAACTCAAAAATAT AACATTGTTTTTCCTTTC T 84 TCTCATACTCAAGAACACAAAA ATGGTGATGATGACCT 85 TCTCCTAAGAAGAAATCAGATAC ATTAACATTTTGAGGGT 86 TCTGCAGAACTAACTCTTTTAA TTTTGCAAAATCAACA 87 TCTGCGGATACATATAATAACTA GAATAAATAACTGTATTTTTT 88 TCTTACCAAAAAATGACG ATTTTCATTAATTTCCTCT 89 TCTGAAGAGCTTACCAAAAC GATAGCTAATTGGTCTGT 90 TCTAGATATACAAATGGAAATTT TAAAAGATGAGCTTCTCG 91 TCTAAAAAAGGACAAGTAAATG AATTTCAATATAGCGACG 92 TCTGATTCTGTCATAAATAAGC CTTGTTTGTCTTTACCTT 93 TCTGAATTTTCACGAGAAA ATTATCCTTCAAAGCTG 94 TACCAATTAGGTAGCTATAA TGTGTCATATAATGTAACCA 95 TCTGTTAATACAAAAACACTTCT TGATCTTAATTTTCGAG 96 TCTGGTCAGTCTAAAAATGAAG CCAAACAGGTTGATCT 97 TCTAGCCAGGAGGTATATG ATTTACATCAGACTGTGAC 98 TCTGAAACTATTAATCCAGAAA TTTATGGCCAATAACA 99 TCTACAAGTATGAACCATCAA TTTTTTAGTAGTTGTCAATT 100 TCTAAGGGGCCAAAAGTAG GTAAGCTGAATTTTCGA 101 TCTATTACTTTAGAAAAATTTAT ACGAGAGTGGTTATTGG AGA 102 TCTGCCTTTTACTTTGGCA TTTCTTCACTCTTTCTAGAG 103 TCTATTTTTTCCTTGATCAT CGGCCAGTTTTTTCTT 104 TCTGGTGAAACCCAAGATA AACACCTGGTGGGCGT 105 TTAACAATTCATGGACC ACTATTTCTAATTGCTCTG 105d TTAACAATTCATGGACC TGGTCCCGGTGCGCCA 105d TCTCAAGGACCTCCCGGTG ACTATTTCTAATTGCTCTG 106 TCTCAAAATCAAAATTCACA CTTAGCAGATTCATCCC 107 TCTCTGGAGCCTTTTATTT TTTACTATTTGAAAATTGG 108 TCTGGTAATCGTTCAGATAAG TTTCATAGGAACTTGTATT 109 TCTATCCAGCAGATCAACT GTCCACACCTGCGACT 109d TCTAAACGGGTTCGCTATG GTCCACACCTGCGACT 110 TCTGTAAAATTAGTATTCGCAC TTTACCTAAGTAATATTCTGA 111.19 TCTGTTAGCGTTGATAAGGC TCCCCGTCTTTTTTGT 112 TCTACAATTAAAAATCTCACTG GTCGTAATCATAAAAGCC 113 TCTAGTAAAATCAAAATTGTAAC TTCATAACGAACCATAAC G 114 TCTAATCTTTTAATTATGGGTT TTTGAGTTCTAGCAACG 115 TTTCAATACTATTTAAAAGG TTTTTTATCTTCTTCTTGC 116 TCTACCGAGGAGCCATTAA TTTTAAAACCTGGTAAAC 117 TCTGAACAATCACAAAAAACA TCAGCTCGTACTGTTT 118 TCTATGGTGACGGTGCTGG GTCCTCCTCAATTGGT 119 TCTAGTCAGCCGGTAGGGG CTCTTTTATACGCGATG 120 TCTGGTGGAGCATTTGCTA GTTATTTGCTCGTTGTT 121 TCTAATAAAGATAATCAAAAAAC TTTCTCAAATGTTTTCAT T 122 TCTGCTGCCACCAAGAAAG TTTCAAATGATCTACAGC 123 TCTACAACAAATGTAATGGC GGCTAGTGTCTGTCCG 124 TCAATGAATTTTTCATTT ACCATCTATTTTTACCCC 125 TCTACAAAATATCAGCGAATG AGAACCCGCACTCTCA 126 TCTACTAAGCAAGCAATGTC GAACGCAACGGCTGCT 127 TCTACAAAAGAATATCAAAATTA TTTCATATCAAAAACTATCG T 128 TCGACTAATTCGTTAAA TTCTTTATCTCTTAATGCTT 129 TTTGAAATAGTATTGGAAA CACAACAGTTATTTTTTCA 130 TCTATATTTTCTATTTTTTATTA AGGCCCTTCTGAGTAG TGT 130d TCTAAAAAACAACTTCACAAC AGGCCCTTCTGAGTAG 131 TCTAAAACAGATATTGAAATAGC AAATAATCCAATGGCTG 132 TCTATTAAATATTATCATTTGCA CTTTTCAAGCTTTTTCC 133 TCTGCTTTACGGAACCTTG AAAATGATCAGTTTGAGG 134 TCTACTATTTCTCAACAACAATA TTTTTGGCTTAAGAAAG C 135 TCTGAAAAAAAGAGTAGTTCAAC CTTACGATACATTTTAAATTG 136 TCTAATCAATTATCAGAAATCA TTCTTTTTTTACTTTAGCG 137 TCTCAAGAGTATAAAACAAAAGA CCATTGCAATCCAGCA G 138 TCTGCTGTATTTACACTCGTC ATGTTTATGGCTTGCT 139 TCTGGCGGCAAGATAAAAT TTTTTGATAAATCCCC 140 TCTGATGGGTTAAAGAATAATG ATATGTGTATTCATCCTTT 141 TCTGATGTTGTAATTAGTGGAG TACTTCTATTTTTCCATCTG 142 TTCGAATTAAGAGAAAGA GTAATGCAATAAATCAAAA 143 TCTAGCTTTTTAGTGATTTCA GGATTTTAGTTTCGCA 144 TATACGCATAGTGGAAC CCCATTGATTTCGTCG 145 TCTGTTATTATCAGGGGCG TACCTCTTTCAATACCAC 146 TCTGTTAGTCGTTCTCCGA ATTACCGTTAGGTACTGTA 147 TCTGAGGAGCAAGAATTAAA GGTATGGTTAACAGAATC 148 TCTATTCTAACAAAAGCAAGT ATATACCCTAGACTTTTTGA 149 TCTAGTGGGCGTTCATGGA AGGAGTTTTATTGATGATAT 150 TCTGATACCCCTAATCAACTA AAATGATTGTGGAAAAA 151 TGCAGGAGCTGTCCGC ATCAAAGAAGTTGACATTG 151 TCTGTCCGCATTGGTAAAG ATCAAAGAAGTTGACATTG Long 152 TCTAACTGCTTAGAAAATGAA GTTAGATAAATTAACCAGTG 153 TCTAACAACTCCAGCA CCCTTTGCTTCGTTGT 154 TCTGGAAAGGTCAGTGCAG TTCCACAAGTCCGATT 155 TCTATTTTATTTTCAGATGAAC TTGTTTGATTCGTCCT 156 TCTGCATCAGATGTTCAGA ACTACCAAACTGCTGG 157 TCTAGTGACGTTGACAAATA TTGTGTATTTTTAGTTAGGT 158 TCTATGACCATTTACTTCAATA GTGGATAAAATTCGAAA 159 TCTCAAACTATTTTGACGC CAGACTGACTAGGAGCT 160 TCTGATGAATATCTACGTGTCG GACTTGTAATTGATTCGC 161 TCTGATGAGGTGGACTATAACA GAAGGCACCACCACCT 162 TCTATTTTCTTGCTCTTAGTTG GTTGTATAGATGAGTTAATCT G 163 TCTGAAACTGTCATTCAACTTG ACGGTTTTTAAAGAATG 164 TATTTTTTAACAACAAAAAA TTTTTCTTTATCTTCTGTG 165 TCTCCAATTTTTATTGGTTT CGATTTTGTAAGAGCTT 166 TCTGCATCTTATACCGTGAA CGACGAAGCTATTTCT 167 TCTACAATTTATATTGCTTGG TAAGGCTTGCATTTTG 168 TCTGTTGGATTGATGTTGG TTTTCCTAAAAATTTTCC 169 TGGAAACAAATCACAG GGCATCTCCTAGCTTT 170 TCTGCAATAGTTTTTACTTTTTT TGATAAAGGTAGTTCTACAC 170d TCTGGTTCTTATCATTTAACAA TGATAAAGGTAGTTCTACAC 171 TCTGCTAGACCCAAACAGT TTTTAGATGTTTTTGTGG 172 TACACTCATATTGTTGAAAA ATGATTGATAATTTTAAGC 173 TCTAATAGTACTGAGACAAGTGC TGCTTTTTGATATGCC 174 TCTGCTTATGTCGTCAATTT TAAAATAAAGTTCAGAAAAG 175 TCTGAATTACCTTCGTTTATC TTTCTCCCTTGACTTTC 176 TCTAAACATCCGATACTTAATG CTTTTTCTCAGATGCTT 177 TCTAATTATCCTTTTGCGA GACATTGAAACGGAAT 178 TCTGGACTACGCGGAGTAT TTTTATCAATGATGTTGA 179 TCTGCTATTGGAGCAGCTG CATATGACGCAAACGC 180 TCTGATAAAGAAGGGATAGAGG AGCCTCTTTTCTTGTT 181 TCTAAAGAAAAATCACAAACTG ACGATTATCAACAAAGTT 182 TCTCAAAATAATAAAAAAGTAAA CATTCTTTTAAATACAAATC A 182d TCTCAAAATAATAAAAAAGTAAA GGGTTTGAAAGTTTTC A 183 TCAAATGGTCAATCTAGC TTTAACTTTAATTACTGGAAT 184 TCTAAGGATTCAAAAATCCC TTTTTTAATAAGCTTCGA 185 TCTGGGCAACCATCTACAT TTTTTTGTAAACTTCCTG 186 TCTCATTCACAGGATAGCA CTTAGATACATTGTTTTTTTC 187 TCTGGACGAGGAGAAGTATC CTTTCTTTTCTTACTTGC 188 TCACAATCTTCTCAAAA TTTATTATTTTTAATACTTGA A 189 TCTGATAAGTCAGCAAACCC CTTCAACTGTTGATAGAGC 191 TCTATCACGACATTACAGACT TCCTTTAGCAGGAGCT 192 TCTAGATATTTAACTGCTGGT GTTATACATGTTGTCTGAAG 193 TCTATAAAATATCAAGATGATTT CCAAATAATAACACGTTT T 194 TTAGAAGTCAGAGAGCAG GCTATCCCTTTCCAAT 195 TCTATTATGGAGACGGGTA TGTATTTTTAATTTGTTTTC 195L TCTTTGAATAATAAAGGTGTCG TGTATTTTTAATTTGTTTTC 195LN TCTTTGAATAATAAAGGTGTCG CAAACTTTTAACATTTAATG 196 TCTATTTCCTCAAATTTTTACG ATAGTGTAAGCTACCAGC 197 TCTAATTTTTATAAGCTCTTG GTCATCATATTCCTGAAA 198 TCTGCGCTTAAAGAATTAA TGTTCGGCGTAAGATT 199 TTTTTAAAAGAAATTGAAA ATTGGTCATTTCTTGAG 200 TTTCGTAAATATAATTTTGA AACAGATTTATTGGTTGG 201 TCTAGCGATACCTTTAATTTT AGACTCATCAACTTTTTCT 202 TCTATGCTGATTAAGTCGC GAACCCTGAAGGGTAG 203 TGTGGTAAAACTGGACT CCAATTGTATTTTTCAAC 204 TCTAAGACAGGAGCACCCGT ATTTATACTACCTGTTGAATC 205 TGCGAGTCAATTGAGC TTTAAATTTGTAGTCTTTAAT A 206 TCTACAAATACTTTGAAAAAAGA CTCTTTTACTTTTCCAAAA 207 TCTAATTTATTTAAACGTTCCT CCCTCCCTTAAGAGAA 208 TCTAAAAAGCGGCTAGTCA TTGACGATGTTGCATC 209 TCTGGACAAAAATCAAAAATA TTTCGAATTATTGTGACT 209d TCTGGACAAAAATCAAAAATA GTATTGTTGTTGCCTG 210 TCTGGAGGAAAATTTCAGAA TTTTTGATTTCCCTTTC 210d TCTACCTCATATCCTTTTATTT TTTATAGTGTGTTTGCAA 211 TGTGGACATCGTGGTG TTTGCTAGGAACTTTGA 212 TCTAAGACTAAAAAAATCATCA TGATTCAATTCCTTTTC 213 TCTAAACACACCAGTAAAGAA TTTTTCCTCTACTTTCTTA 214 TCTAAAAATAAAAAAATCTTATT TTTGCTCACCTCCACA T 215 TTAATAAAAGGATTATTGTCA CAATAACTTCTGTAAAATAAA 216 TCTGCTCGTTTAATACCACA TTCACCCTTAAAATAATT 217 TCTAACACTAACATCCCTAGC TGCATTTTTCCCTTCT 218 TCTAGAGGGAAGGTTATTTAC CTCCAGTAAAGTATTAGTATT T 219 TCTATCAATAAAGTAACAGCTCA GTGAGGTTTTGGTAATT 220 TCTAGAACACTATTTAGAATGAT TGCATATAAGTTTTTTAGC AT 220d TACTATGCGAATCACAG TGCATATAAGTTTTTTAGC 221 TCTAGTTTAGCATTGCAAAT CTCATCTAAAGTGCTATCC 222 TCTACATTTTATAAAAAGACGG CTCGTATTTAGGCAACT 223 TCTAAGAAAATACGAAGCTATAC ATTGGATATGCCATAAA 224 TCTGGAGGAAATGAAATATTA GACTTTTTGATGTTTACTTT 225 TCTGGTATGTCTAATAAGGAAAT TTCTTTACTATAAACATCTTC A 226 TCTAACAAACTTATTACAGAAAA AGCATTTAAAGTTGAATGT 227 TCTGTTTCATATGAAAAAGTCC GTTAGTCTCTTCAAGATCA 228 TCTAGTAGAGGTATTTTTTTACA AAGACCTACCGCCCAA A 229 TCTGAACGTCGGGTAAGTC TACTTCTTTCTCTTTCAATT 230 TTTTTAATCGATTTTATTT CTTAGTGTTCCGATATGA 231 TCATTAATTATTCTTACGGT TCTTGTTTTAAGAGCAGA 231d TCTTTATACGTTGTTAAACA TCTTGTTTTAAGAGCAGA 232 TGGCTAAGTAAGCATGAG ATCATGTTTTCCCTCAA 233 TTCCCAGCTAGCTGTC ATCTGATATATCCGTTTTAT 234 TCTATAGAAATTGCTGTATTAAT TTTTTTGTCTCCTTTTTTA T 235 TCTATTCGATTTCTTATTCTTG AAAGACACGATAAACATAAG 235d TCTGACTCAACCACAGTCTC AAAGACACGATAAACATAAG 236 TCTGCAGACCTTACAAGTCA ATTTGCAACTTCTTGTATA 237 TCTATTGTATTTGCTATTGCA TTTAAAAGTATCCTTAAATAA G 238 TCTGATATTTTTTCAGCTATTGA CTTCCTCCTCAATAGTTG 239 TCTGTTAGTGCTGCTATTGAA TTCTCCTCCCCCATTA 240 TCTAAGAAGCTTACTTTTATTTG ATCCAAACGAGTGAAAT 241 TCAAAAGGATATTCAAGA AGGTGTTGTTGTATTTTC 242 TCTCATAATATATTAAGATTTTT CTTTCTAAGTTTATTAAACAT AGG A 243 TCTATTCTTGGTCAAGATGT GGCATCTGTTACCTTG 244 TCTCATGAAAATGTTAAAAAAG AAACAACTCCATTATTTTT 245 TCTAAGTCAACGGTAACAAA TAAACGTTGAAGAGCAT 246 AGGAAACGTTTTTCCT CTTATCATATCTTGTTAAATC A 246d TCTAACCATAAGGGAAAAGTA CTTATCATATCTTGTTAAATC A 247 TCTGCTAAACAATTAATTGGT TTGCCATGGGTTATAG 248 TCTTTGATGGTGTTGTTATTC AGAATTAAAATTTTCATGC 248d TCTAAAACTTATTTGTCAAATG AGAATTAAAATTTTCATGC 249 TGGGCTTACCATACTG TTTTTTAGATGTTTTATGTG 250 TCTGGCCTTAATCTTAAGC CTCTTTTACTTTAGCTTCA 251 TCTCAATATTTTTTGAAACAAG TTTCAAACTCCAGCCA 252 TTTATTTCAGGTTATATCAA GGAGTGCCTTTCTACT 253 TCTGAAAATTGGAAGTTTGC TTCATATCGTAAAGCATC 254 TCTATTGAAAAGGGAGTTG ATCGTCAACCTTAACG 255 TCTATTGTTGGTAGAGAAATCA TTTTACTTGACGTCTCAC 256 TATCATGTAAAAATTGATCA GTCTTCCATTAATATTCCC 257 TCTGATTTTTTATACAAAGGAGG CCAATTATTTTGAAAGTTC 258 TCTGAACGTTATACAGATAAAAT ATTTTTTTGAATAATATAATC G C 259 TCTCTTTCTCGTAAAAAAGAG TTTATTATCAGAAAAGGC 260 TCTACTCTTGTCTTAGTTGTTTA ATTCAAAAAATTTTTCAA T 261 TCTATAAAGAAAGCTGAAAATC CGAAACGTCAGGTAAA 262 TCTATAAAAAATGCTATAGCATA ACTTATTTTTGATAATATTTC TT 263 TCTCAGCCTTCTAAACTACTTC ATCAGCATTTCTACGAA 264 TCTGATTTGTTTAGCATGTTG ATGTAGACTCCTAATGATTT 265 TCTCTTGCTTCCCTGATTT TTTACTGTTCCTTTCGC 266 TCTCATCAATCAAATCATTATC GAGATTAATTTGATTATATTT T 267 TCTATCTTTATTATCGGACAA AACATCATTTCCTCCC 268 TCTAAAGAATTTATTAAAGAATG GTTGATAGTTCCAAAACG G 269 TCTGCAGATGATGGTGGTT TAAATGTGTTCCTACTAAATT 270 TTAAATGATGCAATAACAA CATCAATAGCCGAGCTG 271 TTGCTGGATTATCCTC TTTATTTTCCAAATGACA 272 TCTGTATTTATGGCAAATAAGA TTCACTCGGAGTTGGAG 272d TCTATGAGTTCTCTGGAAGTT TTCACTCGGAGTTGGAG 273 TCTGGTGTCCTCAACTCTG AATGTAAATGACAAAGGTA 274 TCTGTTCATGATTTTGGTGA GTTTTTTAATGGTTTGC 275 TCTGGGGTTTGGTTTTATA TTTATCATAAGCATCTAGAC 276 TCTCAATCAGACATTAAAGCA CTGATCTCTTGTTGATGC 277 TCTATTTGGAGGGGGGAAA AAGCAGGGGAGCAATA 277d TCTACCAAATTTGACTGGG AAGCAGGGGAGCAATA 278 TCTGTTACGTTTTTCTTAT CTGAGCAACACCTGTC 279 TCTAAAAAGAAAAGTTTAATTAG GGCAATTTTGTGGCAA C 280 TTTGATTTTTTTAAGAAAA TTGCTTAGTTAATGGCT 281 TCTAAGAAATTAATTATAGGTAT AGGCGTTGAATATAATTC TT 281d TCTGGTTTTTCGTTTTTGA AGGCGTTGAATATAATTC 282 TCTCTATTCTCAGATGAAACAA CTTTTCAACTCCAAACA 283 TCTGTTAAATTAAAATCGTTACT GAGTTGTCTTTTTTTGTC G 284 TCTATGCAACGATTAGGAC GCAATCACAATTGACAT 285 TTAGGTGAAAGCAAATC CTTTGTCTGCTTCACTT 286 TCTGGAGGATTTTATATGAAAG TTGTATCTTCTCCTGACC 287 TCTGCACACACACCTACTAGT TTGGTTAATCGTCTTG 287d TCTAACAATCGTTCAAAGC TTGGTTAATCGTCTTG 288 TCTAAAAAGTTTTTAAAAGTTTT TTTAGTTACTTTCATAAATGG 288d TGGAATAATCATCAGTCA TTTAGTTACTTTCATAAATGG 289 TCTCAATCTAAAGGGCAAA ATATAATTCCTCTAAAACTAG C 289L TCTCAATCTAAAGGGCAAA CCACTTCAAATTAACTAAC 290 TATTACTTATCAAAAGAAAAGG ATTCCTTGAACACGAA 291 TCTCAAGTATTAAATGACAATGG GTGCCATTCATTCTCT 292 TTGAATCGTAAAAAAAGG TTGTCCTGTGAACTGTG 293 TCTATGGGTCTAGCAACAA AGGGTTTATTTGTTGAAG 293d TCTATGGGTCTAGCAACAA TCCTGATTTATCCACTG N-term 293d TCTGTTACAGCTAAACACGG AGGGTTTATTTGTTGAAG C-term 294 TCTGGTCATTTTAGTGAAAAA CAAAATACCTAAGCTAGC 295 TCTAGCGACATAAAAATCAT ACGAACTTCCATAACC 296 TCTAAAGGTATTATTTTAGCG GGCTTCTCCAATCAAA 297 TCTATTCAGATTGGCAAATT TTGAGTTAATGGATTGTT 298 TCTACTAAATTTATTGTTGATTC TAGCGTTATTTCACTGTG A 299 TTTGAAATACTTAAACCTG TTTCTCCGCCCAGTCA 300 TCTGCTTCTACAAATAATGTTTC CCGTTTATTCTTTCTACTG 301 TCTGTAATTAATATTGAGCAAGC CATATCTGTTGCATCAAT 302 TCTGAAATCAACACTGAAATAG AACTGGCTTTTTAGTCAG 303 TCTACAAGGCATATAAAAATTTC TTTATTATTTAATTCTTCAAT A 304 TCTAACGAAATCAAATGCCC GTCTTTTAGAGCATCGA 305 TCTGGACGAGTAATGAAAACA CTCTCCTCTAAGACTTTCG 306 TCTGGGAAAAAAATTGTTTT TCCTTTTGTTACTTTTGC 307 TCTAAATTTACAGAACTTAACTT TTTATCGCCTTTGTTG AT 308 ATGACACAGATGAATTTTA ATGTTCAGGTTCTCCG 309 TTGCAACTTGGAATTG TTCCATTATCTTCAAGTTA 310 TCTGCTAAAGAGAGGGTAGAT CTCTTCTTCATTTTTCTTA 311 TCAATTATTACTGATGTTTAC TTTTTTTAAGTTGTAGAATG 312 TCTACTGCAACTAAACAACAT GTTTTTTGATGCTTCTTG 313 TCTAAACGTATTGCTGTTTTA TTTACTACTTTGGTTGGC 314 TCTAAATTTTATCTTGTTAGACA GTGTGTCATTTTGACCT C 315 TCTATAGGGGATTATTCAGTAA TCCTTCAAGATCATTTAA 316 TCTACTGAACGAACATTCGA ACCTCCTTTTCTTTCATT 317 TCTAATAAGCCATATTCAATAG ATCTTCTCCTAACTTACCC 317d TCTAATAAGCCATATTCAATAG ACTAGCTAGATTCTTAACGC N-term 317d TCTGACTTGAATGGCAATAT ATCTTCTCCTAACTTACCC C-term 318 TCTATTGATTTTATTATTTCTAT GCCTCTTTCTCCAAAT TG 319 TTAAAACATTTTGGTAGTAA ATGTCCTGTTATATCTTCTT 320 TCTACTATTTATGACCAAATTG GCGTTGAATAATGGTT 321 TCTAAAAATAAAAAAGATCAGTT TATTTCTTTAGTTTCTTCAA 322 TCTCAAGAAACAGATACGACG TAATAAAAATTATATAAGAAC CT 323 TCTGGTAATGAGTCAAAGAAC TTCTGTCTTATAAGCATAAG 324 TCTGGAAGTAAATCAGCTTC TTTTTTATAAGCATGTGTA 325 TCTGCTTGGCAACTTGTTC ATGAGACATAAGGTCTTG 326 TCTGGCATCTCAGACTTACC GTTGGAGCTCCTACTG 326L TCTAAATTCAAATCTGGGG GTTGGAGCTCCTACTG 326L TCTAAATTCAAATCTGGGG CATTTCTTTGGTTAAAGC N-term 327 TCTGGAGGGAAAATGAATC TATCTCGAGTGCTATTTG 327d TCTGGAGGGAAAATGAATC CTCTTCATCGACATAGTAA N-term 327d TCTGGCAACTTCAAAGCAT TATCTCGAGTGCTATTTG C-term 328 TCTGACCAAGTCGGTGTCC ATTTTACAGTAGTGGAGTTT 329 TCTAAATCAAAGACCTCTTCTA TGTCCTCATTTTTTCA 330 TCTAATAAACGCGTAAAAATC TTTAACAGTACGAACACG 331 TCTACCAGAACAGTAGCAAT CCCCCTGTTTTTAAAAT 332 TCTACAAAAAACCTGTTATTAA ACCCTCATATGATTCC 333 TCTATTGATATACAAAAAATAAA TTTAAAATAATGATACATCTC A 333d TCTGGATCATTGAGGGCAA TTTAAAATAATGATACATCTC 334 TCTAATTTAGTAAAAGTGAATAG TAACCCCGTCTCAACA TG 335 TCTGAAGAAGAAAAATATTTTGA TATTTTCGTTTTCTCAAA 336 TCTCAGGTTGAAGTTGACTTA TTTCTCCAAATAATCTCTC 337 TCTGAAACAGATTCGTTTGTA CCTACTTTTAGTTTTAGAAGA 338 TCTGCTATAATAGACAAAAAG GAAATCATAGCTTCCC 339 TCGAAACCGATTAAGAT ACCTTTTACTTTTGGTAGT 339d TCTCAAGTCATGCGCTATG ACCTTTTACTTTTGGTAGT 340 TCTGGATTTCTCTATAATTACTT TTGTTTGTGAAGTAAAACG C 341 TCTGGAAAACCATTGTTAAC TAATTTAAAAATTGCATAAA 342 TCTCAGAAAATTGAAGGTATT TTTCGTTACCATATCTAGA 343 TCTGAAATGCAAGTTCAAA TAAATCATGGAAACTAGC 344 TCTGCACAACGCAGAATGT AAAGCCCAACCTTCCG 345 TCTAAAAACCTGAATTGGG GTTTCCACGTCCTTTC 346 TCTAATAAAATAGCTAATACAGA AAGTTTATTCAAATCTGG AG 347 TCTATTGATATTCATTCTCATAT AATGTAATGGTTTTTTAATA C 348 TCTACTGGATCTAAAAAATTAGC AGCTAAAATACCTAACCAG 349 TCTAAAGATCGCTTATATAATAA ATTTTTTAAACGACTCAT A 350 TCTGCAAAAGATATAATTAAGGT AGCGGAACGGTGAATA T 351 TCAGAAGATCAAAAACA ATAATCTAAACTATCAGCTCT 352 TCTACTTTTTTTAAAAAGCTAAA ATCTCCTATTGTAATTTTGA 352d TCTGGTACAGATAGTAAATTTGG ATCTCCTATTGTAATTTTGA 353 TCTACAATGTTAAAAATTGAAA CACCTCTTTTGTCAGA 354 TCTATTAAAGAACTAAAAGAATT TTTGTTAGCGAGTAAGTC T 355 TCTCGCTCACTACCTT TTTATCATCCTCCTTAATAA 356 TCTAAATTCTATATTATTGATGA AAACGTTTTACTCTGTAAAA TG 357 TTGGAACATTTTTATATTAT AAATAAGAATGTTAAAAGAGC 358 TTTTATACAATTGAAGAGC TTCCCCAAAAATTTCT 359 TCAAGAAATAATTACGGT ACGCAGTCCCATTTTC 360 TCTATAATGAAGGCGGTCT CTGGCATGAGGTCTCA 361 TCTAGCGTATATGTTAGTGGA CCTTTTTTCAATAATAGC 362 TCTACTAAACCACAGGGGG ATCTTTAATCTTACCATCC 362d TCTACTAAACCACAGGGGG TGCTGCTACTGCAATG N-term 362 TCTGGTAATGAAGGAAATATCAC ATCTTTAATCTTACCATCC C-term 363 TCTCTCGAATTAAAAAATATTG TAAATTCCTTTGTTGTAATA 364 TCTAACTATATGGGTATGGGC ACCATCAGTTGTCACC 365 TCTGGAACTGCTACATATAGTAG TATTGACCAGTGCACG G 366 TGGCTTGACATTATTTT TTTTTTTGAATTTGTAAAAG 367 TCTAAGAAATTAAAAATATTCCC AGAGATTATTTTTATTTTAAA T 368 TCTAAAATCATTATTCAACGT TTTATTTTTAGTATCTAAAAC G 369 TCTAGTAGAATGATTCCAGG TTTAGAAACTCCAAGTATCTC 370 TCTACCGAATTTAATGACG GTTAATTTGACTATTGATATA TT 371 TCTAAAGATAGATATATTTTAGC TAAACTCTCAAAAGCTAAAC AG 372 TCAGAAAAATATTCCACT ACGTTCTTCTCTGGCT 373 TCTGAAATTGGTCAGCAAA ACTTAAATGGAACAACC 374 TCTAAGTTCGAAAATATAATATA TTTGCCTAAAAAATTAGG TG 375 TCTGAAAAAGAAACTATTTTAAG GGCTTTCCTCCCTTCA T 376 TCTAAAGAAAAGAAAAATTTGG TTCATCTTTTTCAATATCA 377 TCTGGTAATAAACTGATGTATCA GTGAGAGTGTCTTTGTTT 378 TCTGAAGATCAACTCACTATATT CAGATTTTTAGCTACTTGTC T 379 TCTCAAATTACCCGAGAAG TCTAGAGCGCTTTATAAG 380 TCTCTTAAAAGATTACTTACTGA TTTTCTAATAGTTAGAAGCC AG 381 TCTCTTGGGATAGCTCACA TTTTAAATGTGCAGAGA 382 TCTATAAAGTTTAAATTATTTTT ATTTATAATTTCCTTGGG TAA 383 TCTATTTTACAGACGAATATACT TCTATAATATCTCTCTAAAGT AT GA 384 TCTAGAATAATTGTTGTCGG CCTCGCTAACATATCAC 385 TCTAATGTAAAAAAACGC AGCTCTTACAGTCTTGC 386 TCTCTAGTATCAAAGGAGAAAGC TTGTCTGAGTGACCAA 387 TCTGGTATGTTGTTAGCA ATAATATGAAATATGTTGTTC A 388 TCTCTTATGATAATAAATTCATT TCCGCAGAGTAAAAAA CG 389 TCTATGAATAGTGAACATAAAAT TTCATAAATGTGCCAA T 390 TCTAGGGAAACTTACTGGA TTCATCTCTGCTCACC 391 TCTAAAAAAGTCATCGATTTAA TTCTCCTTCAGCTTTTA 392 TCTATTACATATGATTTCACAAG GTCATTTTTTCTAAAGTTTG 393 TCTAATAAATCTTGGTTGAGAA TTTTTGTAGTTGTTTCAAT 394 TCTCCTATGTTGTCTGTTGG TTTCATTAGATAACTATTCAG C 395 TCTACTTATCAAAAAACAGTTG TATAGACTGAAGATAATTAAT TAA 396 TTTGTCAAAGGGATTT AAATCGATTAATCAAGTC 397 TCTAAATTATTTGATAAGTTTAT TCTAAAGTAGTCCTTTAGACT AGA A 397d TCTAAAACTGCTACAGTTAG TCTAAAGTAGTCCTTTAGACT A 398 TATTTAGAACAATTAAAAGAGG TTTGTCCATAATCATTTC 399 TCTAAAGTTTTAGTAGTTGATGA GGTAGATATGCCTAACATT T 400 TCTAAAATAGTTGAAGGCG GTTTCCTTCCAAAAAA 401 TCTGGAATTGAATTTAAAAATG TCCATGCTTAATAGCC 402 TCTGGAAAATATTTTGGTACAG ATCTAAACCAATTTCTGTAC 403 TCTGAGGTTAGAATGGTAACTC GTCCACAAAAACGTCT 404 TCTAAAATAGATGACCTAAGAAA TAGATGTTCTACGGAGAA 405 TTGAAAATTCAGTATTATCA AAAGATGGCAAGCCAT 406 TCTGATAAAAATAATTTAGAAGA TCTCTCTCCACACCATA CT 407 TCTAAAATTGACATGAGGAA CTTACCTCCTGTGGCT 407d TCTAAAATTGACATGAGGAA CTTTTGTTGGTTACCTC 408 TCTAACCACTTACTTAACCTCA TATTGTTAAATATGATGAAAT G 409 TCTAAGGTAGTAGTAGCTATTGA ATGATTATACAAATTGATTAA T T 409d TCTACTGAAGAGAGAAATCCT ATGATTATACAAATTGATTAA T 410 TCTGCTTTATTATCAGTTATTGT TCCCTCTTCCTTGACA C 411 TCTAAAGACTATATTAACAGAAT AACGTTTTTGAGCTTT ATT 412 TCTGGATTTTTTGCACAGC TTTTGTCTTAAACGTTCT 413 TCTATTGTTGGTGAACAAGA TTTAGATAGTCTAGCCATTT 414 TTAAATCAATATTTTCTGC ACGGCTTGGGGCAGAG 415 TCTGAGCGAATTCCTGTTC TACCATTATCCGTGCT 416 TCTGAAGTCATTCGTGAACA ACTATTAAACTCCAATGTTA 417 TCAAAACAATATGATTATATC GCGCATTGTAACAAAT 418 TCTAGCAAGCCTAATGTTG TTTTGGTAAAAGGTCTG 419 TCTGATTTAAATAATTACATCGC TCCTGGAAAGTTCATC 420 TCTAAACGTGAATTACTACTCG TAGTTTATCTAAAGCGTTC 421 TCTATACGCCAGTTTTTAAG TTTATGTATAGAAACAGCAG 422 TTTTCGAGCGATTTTG AATGTACATAACAATAGAGAG C 423 TCTGTAACCAAAGTTGAAGAG CAACGATCCCAAGAAC 424 TCTATGAAAGATTTTATTGAATG GCCATTCTTACCTCCT 424d TCTATGAAAGATTTTATTGAATG ACGTTTTTTCTGACCG 425 TCTATAGCCTTTAATAGTTTATT TATAAAATAAATTTGAAGATC T T 426 TCTD440ACAGTTTATAATATAA ATCATCTTGTACCAACTC ACCATG 427 TATTCTTTTGAAGAACTTTT GCCAATAAATTCACGG 428 TCTATAAAAATTTTGATCCC AGTCTGTTTTTTAACAAAAG 429 TCTAATCATTCCATTGAATC TGGTTTTAGAACAACTTTA 430 TTACAAAAAAAATATCGG AATTAAGCTGAAAATGAC 431 TCTGCGGCTCAATTAGCTG ATTATATTCTTTTAATTTGTC A 432 TCTCGTACCTTCAAACCAG CTTACGACGTCCTGGA 433 TCTATTAAAGCAACTTTTACTC GTGTGTCATGACTACTGTAC 434 TCAATTTTTCAGACAACA TGAGTAGAGCACAAGC 642 TCTAGAAAACGTAATGATACATT GAAACGAATACGTTCTT 643 TCTGATTGTCAAATTACACCA ACTACCTACCGTTTTCAC 644 TCTATTTTTCGTGGTGATAA TTTGATGGTAACAGTCG 645 TTTTTTAATATTGAATATCAC AGAAAGGCGCTCTTCT 646 TCTAAGGGAGTCCAATATATG TATCTTTAATAAAGCCCTA 647 TCTCGTCGCATGAATACCA CATCCCATAAATTTGTT 648 TCTATAGAATTTTCAGGGC CAAGACATTTCTTAAAGC 649 TCTGCTACTCACTCTAACTCAG TTTTGTTTTAGCGATG 650 TGCTCTTCTTCAAATACT TTTTAAACCATGCTGT 651 TCTCTAACACCATTTACAAAAG TTTGTAAAGACCTTCTTT 652 TCTCAACAAGGTATTATGGATA TTCCTCGTTTATTAATTT 653 TCTAAAATTTTAGGTACACCA AAAGAAAAGATGTGCC 654 TCTGGAAAAATGGTTAAGAA CTGTGCAGGCTCAAAT 655 TCTAAATTCGTCCGAACCGT AATTGTCCAGTCTAAGTTA 656 TCTGGTCTTCCAACGCAGC ATTTAGTGTTATTTCTCCTG 657 TGCTCAGGTAAAACAT TTTTTTAAGTGATGATGAA 658 TCTGAAAGCAAATCTTTGC CTTTGTCTGCTTCACTT 659 TGTGCTAATTGGATTG TTTTGGGGTTACTTTAC 660 TGTGGAAATGTCGGAG TTTTGCTGAAATAATGTT 661 TGTCAGTCAAACCACA ATCATACGAATGCAAC 662 TCTGCTAGTTTTTATTTTTTCC TTTTTCATATTTTTTCAAA 663 TGTGGAAGTAAATCAGC ATTATTTTTATAAGCATGTG 664 TCTGTTAAATTAAAATCGTTACT GAGTTGTCTTTTTTTGTC G 665 TCTATTGCTGGTCCTAGTG GATAAGCACTTTCCTTAA 666 TTATTTTTTGGAAATTGG GCCTAAAAACCAATCA 667 TCTGCTGTATTTACACTCGTC ATGTTTATGGCTTGCT 668 TTTTATATGAAAGAACAACA TTGTATCTTCTCCTGACC 669 TCAATTATTATTGGGTTAA ATATACCCTAGACTTTTTGA 670 TCTCCTAAATTAACCCTAGTCT GGCTTTAAAGTTCGATA 671 TCTAGTCTTGCGAAGGCAG TTTATCGTAAGCACTTAGG 672 TCTGTATTTACACTCGTCTTACA ATGTTTATGGCTTGCTT 673 TCTGGAGGATTTTATATGAAAG TTGTATCTTCTCCTGACC 674 TCTGTTAAATTAAAATCGTTACT GAGTTGTCTTTTTTTGTCT G 675 TCTGGTTCATCAGACAAACA TTCAACTTGATTGCCA 676 TCTGTAGTTAAAGTTGGTATTAA TTTTGCAATTTTTGC CG 677 TCTGTATTAGAAGTACATGCTGA TTTTAATGCTGTTTGAA 678 TCTGAGACACCAGTAATGGC TTTTTTAGCTAAGGCTG 679 TCTGCTAACAAGCAGGATC TTTTGCTAAACCTTCTG 680 TCTAATAAGTCCAGTAACTCTAA ATTCATATTAACACGATGC G 681 TCTGCTTTTGATGTAATTATGC TTTGCGTTTTGGAGGG 682 TCTATTAACTATGAGGTTAAAGC TGCACCTTGATGGCGA 683 TCTGTAATTGTTGAACTTAGTTT CCATAATATTTGATGCTG G 684 TCTCTTAGGAAGTATAAGCAAA TTCTAATCCTACAGCATG 685 TCTAAAATTTGTCTGGTTGG AAAAATTCCTCCTAAATTAA 686 TCTGACTTTTATGATATCAATCT AAAGTTTTGACTATTACTGAT T AG 687 TATGCTATTATGCAAAAAG TGGGGGAGATAGTTATG 688 TCTGCAATCGTTTCAGCAG TTGACAGAAAGCTAATTG

TABLE III RESULTS FOR in vivo GBS CHALLENGE % survival GBS # Pre-immune Post-immune  1 18.7 22.2  4gst 19.4 37.2  4his 25.0 75.0  8 14.3 42.1  10 29.1 36.0  15 30.0 60.9  16 33.3 53.8  18 29.4 50.0  21 5.9 10.0  22 36.8 63.1  24 38.5 41.4  25 28.6 85.7  32 20.0 25.0  35 0.0 17.6  45 26.7 37.5  48 20.0 25.0  52 14.2 17.3  53 23.8 29.2  54 22.7 44.0  55 50.0 52.9  57 33.3 55.6  58 6.7 11.8  62 15.8 36.4  63 21.4 42.9  65 3.7 23.3  67 23.5 27.8  71 13.3 26.7  73 28.6 39.1  80 38.8 56.5  84 33.3 37.5  85 30.8 62.5  90 14.3 22.7  94 25.0 30.0  95 16.7 23.1  98 5.9 11.1 100 26.9 42.9 103 16.7 52.9 106 10.0 18.2 110 11.1 30.0 113 17.6 29.4 114 40.0 52.2 117 27.8 36.8 119 36.4 52.2 139 23.1 26.7 150 21.6 44.4 153 25.0 30.0 155 22.6 36.8 157 14.3 31.8 158 22.6 40.0 163 29.6 37.9 164 25.0 43.8 173 17.9 38.7 176 20.0 38.9 177 21.7 33.3 181 5.0 21.7 186 41.2 52.6 188 11.8 23.5 189 21.4 31.6 195 32.1 64.7 206 33.3 50.0 211 30.8 33.3 232 50.0 57.1 233 34.8 55.2 236 57.1 70.6 243 46.7 52.9 263 15.4 35.7 273 61.5 75.0 276 23.8 44.4 296 25.0 28.6 297 13.3 23.5 298 20.0 22.2 302 30.0 52.2 304 33.3 40.9 305 42.1 70.0 316 38.5 42.9 318 7.1 15.8

TABLE IV COMPARISON OF GBSnnn NUMBERING AND SEQ ID NUMBER GBS numbering Sequence listing GBS1 SEQ ID 3532 & 8736 GBS2 SEQ ID 4530 & 8818 GBS3 SEQ ID 6266 & 8958 GBS4 SEQ ID 2 & 8786 GBS5 SEQ ID 2598 & 8674 GBS6 SEQ ID 398 & 8496 GBS7 SEQ ID 8790 & 9798 GBS8 SEQ ID 8694 GBS9 SEQ ID 4540 & 8822 GBS10 SEQ ID 8718 GBS11 SEQ ID 5884 & 8930 GBS12 SEQ ID 8764 & 9692 GBS13 SEQ ID 8484 GBS14 SEQ ID 5406 & 8892 GBS15 SEQ ID 4 & 8710 GBS16 SEQ ID 944 & 8538 GBS17 SEQ ID 1770 & 8602 GBS18 SEQ ID 6860 & 9002 GBS19 SEQ ID 4422 & 8812 GBS20 SEQ ID 308 & 8488 GBS21 SEQ ID 8762 GBS22 SEQ ID 8584 GBS23 SEQ ID 8512 GBS24 SEQ ID 1694 & 8598 GBS25 SEQ ID 3180 & 8714 GBS26 SEQ ID 8820 GBS27 SEQ ID 8774 GBS28 SEQ ID 8738 GBS29 SEQ ID 8744 GBS30 SEQ ID 8860 GBS31 SEQ ID 8702 GBS32 SEQ ID 8910 & 10142 GBS33 SEQ ID 5734 & 8912 GBS34 SEQ ID 5750 & 8916 GBS35 SEQ ID 8908 GBS36 SEQ ID 8542 GBS37 SEQ ID 8564 GBS38 SEQ ID 2122 & 8642 GBS39 SEQ ID 8480 GBS40 SEQ ID 8654 GBS41 SEQ ID 1176 & 8562 GBS42 SEQ ID 4856 & 8850 GBS43 SEQ ID 672 & 8520 GBS44 SEQ ID 9000 GBS45 SEQ ID 9018 GBS46 SEQ ID 1834 & 8608 GBS47 SEQ ID 8588 GBS48 SEQ ID 8594 & 8596 GBS49 SEQ ID 8494 & 9490 GBS50 SEQ ID 1236 & 8566 GBS51 SEQ ID 5410 GBS52 SEQ ID 3920 GBS53 SEQ ID 8586 GBS54 SEQ ID 3442 GBS55 SEQ ID 9020 & 10338 GBS56 SEQ ID 2510 & 8668 GBS57 SEQ ID 8854 GBS58 SEQ ID 8664 GBS59 SEQ ID 3744 GBS60 SEQ ID 8760 GBS61 SEQ ID 8776 GBS62 SEQ ID 2244 GBS63 SEQ ID 390 GBS64 SEQ ID 374 GBS65 SEQ ID 8544 GBS66 SEQ ID 3028 GBS67 SEQ ID 3746 GBS68 SEQ ID 4012 GBS69 SEQ ID 4916 GBS70 SEQ ID 3718 GBS71 SEQ ID 8906 GBS72 SEQ ID 1348 GBS73 SEQ ID 220 GBS74 SEQ ID 5872 GBS75 SEQ ID 8926 GBS76 SEQ ID 5862 GBS77 SEQ ID 3256 GBS78 SEQ ID 3262 GBS79 SEQ ID 3264 GBS80 SEQ ID 8780 GBS81 SEQ ID 2706 GBS82 SEQ ID 2898 GBS83 SEQ ID 8772 GBS84 SEQ ID 4182 GBS85 SEQ ID 216 GBS86 SEQ ID 2978 GBS87 SEQ ID 3452 GBS88 SEQ ID 5694 GBS89 SEQ ID 2682 GBS90 SEQ ID 8476 GBS91 SEQ ID 8938 GBS92 SEQ ID 8964 & 10238 GBS93 SEQ ID 2848 GBS94 SEQ ID 1592 GBS95 SEQ ID 2224 GBS96 SEQ ID 2130 GBS97 SEQ ID 800 GBS98 SEQ ID 8746 GBS99 SEQ ID 4240 GBS100 SEQ ID 8782 GBS101 SEQ ID 6902 GBS102 SEQ ID 6894 GBS103 SEQ ID 6 GBS104 SEQ ID 8778 GBS105 SEQ ID 1400 GBS106 SEQ ID 8502 GBS107 SEQ ID 6026 GBS108 SEQ ID 8532 GBS109 SEQ ID 4116 GBS110 SEQ ID 6832 GBS111 SEQ ID 8842 GBS112 SEQ ID 8904 GBS113 SEQ ID 300 GBS114 SEQ ID 8968 GBS115 SEQ ID 5164 GBS116 SEQ ID 5152 GBS117 SEQ ID 8962 GBS118 SEQ ID 2508 GBS119 SEQ ID 8814 GBS120 SEQ ID 8874 GBS121 SEQ ID 3826 GBS122 SEQ ID 9006 GBS123 SEQ ID 6310 GBS124 SEQ ID 260 GBS125 SEQ ID 3872 GBS126 SEQ ID 6736 GBS127 SEQ ID 8816 GBS128 SEQ ID 752 GBS129 SEQ ID 8990 GBS130 SEQ ID 9004 GBS131 SEQ ID 6198 GBS132 SEQ ID 8730 GBS133 SEQ ID 474 GBS134 SEQ ID 9008 GBS135 SEQ ID 8882 GBS136 SEQ ID 1188 GBS137 SEQ ID 3960 GBS138 SEQ ID 9052 GBS139 SEQ ID 884 GBS140 SEQ ID 8632 GBS141 SEQ ID 1768 GBS142 SEQ ID 8600 GBS143 SEQ ID 9054 GBS144 SEQ ID 2238 GBS145 SEQ ID 8700 GBS146 SEQ ID 8696 GBS147 SEQ ID 8526 GBS148 SEQ ID 9010 GBS149 SEQ ID 8732 GBS150 SEQ ID 3736 GBS151 SEQ ID 3188 GBS152 SEQ ID 3952 GBS153 SEQ ID 3904 GBS154 SEQ ID 4024 GBS155 SEQ ID 8796 GBS156 SEQ ID 4646 GBS157 SEQ ID 4812 GBS158 SEQ ID 5504 GBS159 SEQ ID 8628 GBS160 SEQ ID 8924 GBS161 SEQ ID 8922 GBS162 SEQ ID 168 GBS163 SEQ ID 224 GBS164 SEQ ID 1102 GBS165 SEQ ID 3672 GBS166 SEQ ID 8712 GBS167 SEQ ID 4214 GBS168 SEQ ID 9016 GBS169 SEQ ID 4346 GBS170 SEQ ID 8982 GBS171 SEQ ID 6720 GBS172 SEQ ID 6704 GBS173 SEQ ID 8788 GBS174 SEQ ID 6150 GBS175 SEQ ID 62 GBS176 SEQ ID 8478 GBS177 SEQ ID 8876 GBS178 SEQ ID 6078 GBS179 SEQ ID 8848 GBS180 SEQ ID 3062 GBS181 SEQ ID 1924 GBS182 SEQ ID 3774 GBS183 SEQ ID 4796 GBS184 SEQ ID 1978 GBS185 SEQ ID 1046 GBS186 SEQ ID 8470 GBS187 SEQ ID 844 GBS188 SEQ ID 3410 GBS189 SEQ ID 6986 GBS190 SEQ ID 8842 GBS191 SEQ ID 1814 GBS192 SEQ ID 8618 GBS193 SEQ ID 2382 GBS194 SEQ ID 3912 GBS195 SEQ ID 8 GBS196 SEQ ID 4944 GBS197 SEQ ID 5486 GBS198 SEQ ID 8896 GBS199 SEQ ID 1162 GBS200 SEQ ID 8936 GBS201 SEQ ID 4550 GBS202 SEQ ID 8666 GBS203 SEQ ID 6478 GBS204 SEQ ID 1996 GBS205 SEQ ID 18 GBS206 SEQ ID 8552 GBS207 SEQ ID 3822 GBS208 SEQ ID 3916 GBS209 SEQ ID 3918 GBS210 SEQ ID 3738 GBS211 SEQ ID 4680 GBS212 SEQ ID 8750 GBS213 SEQ ID 8500 GBS214 SEQ ID 8498 GBS215 SEQ ID 9022 GBS216 SEQ ID 8606 GBS217 SEQ ID 9024 GBS218 SEQ ID 8652 GBS219 SEQ ID 8646 GBS220 SEQ ID 2730 GBS221 SEQ ID 9028 GBS222 SEQ ID 3842 GBS223 SEQ ID 8794 GBS224 SEQ ID 9026 GBS225 SEQ ID 8834 GBS226 SEQ ID 4966 GBS227 SEQ ID 5030 GBS228 SEQ ID 5050 GBS229 SEQ ID 9056 GBS230 SEQ ID 1296 GBS231 SEQ ID 5810 GBS232 SEQ ID 5830 GBS233 SEQ ID 4722 GBS234 SEQ ID 1106 GBS235 SEQ ID 8560 GBS236 SEQ ID 6162 GBS237 SEQ ID 8706 GBS238 SEQ ID 4246 GBS239 SEQ ID 8980 GBS240 SEQ ID 8986 GBS241 SEQ ID 9030 GBS242 SEQ ID 9032 GBS243 SEQ ID 8678 GBS244 SEQ ID 6554 GBS245 SEQ ID 8994 GBS246 SEQ ID 6864 GBS247 SEQ ID 8856 GBS248 SEQ ID 454 GBS249 SEQ ID 8620 GBS250 SEQ ID 8634 GBS251 SEQ ID 2258 GBS252 SEQ ID 8648 GBS253 SEQ ID 2526 GBS254 SEQ ID 2710 GBS255 SEQ ID 2966 GBS256 SEQ ID 3424 GBS257 SEQ ID 3550 GBS258 SEQ ID 3752 GBS259 SEQ ID 8756 GBS260 SEQ ID 4162 GBS261 SEQ ID 1530 GBS262 SEQ ID 8572 GBS263 SEQ ID 1616 GBS264 SEQ ID 8824 GBS265 SEQ ID 4554 GBS266 SEQ ID 4652 GBS267 SEQ ID 4980 GBS268 SEQ ID 5038 GBS269 SEQ ID 5534 GBS270 SEQ ID 1998 GBS271 SEQ ID 8570 GBS272 SEQ ID 22 GBS273 SEQ ID 5994 GBS274 SEQ ID 774 GBS275 SEQ ID 2308 GBS276 SEQ ID 8942 GBS277 SEQ ID 8954 GBS278 SEQ ID 8524 GBS279 SEQ ID 6292 GBS280 SEQ ID 6254 GBS281 SEQ ID 4458 GBS282 SEQ ID 4444 GBS283 SEQ ID 9034 GBS284 SEQ ID 6456 & 8974 GBS285 SEQ ID 8802 GBS286 SEQ ID 9036 GBS287 SEQ ID 5354 GBS288 SEQ ID 5374 GBS289 SEQ ID 8616 GBS290 SEQ ID 8680 GBS291 SEQ ID 8530 GBS292 SEQ ID 8998 GBS293 SEQ ID 8582 GBS294 SEQ ID 8604 GBS295 SEQ ID 2722 GBS296 SEQ ID 2658 GBS297 SEQ ID 3024 GBS298 SEQ ID 8704 GBS299 SEQ ID 3268 GBS300 SEQ ID 4170 GBS301 SEQ ID 8576 GBS302 SEQ ID 8670 GBS303 SEQ ID 8554 GBS304 SEQ ID 5846 GBS305 SEQ ID 208 GBS306 SEQ ID 212 GBS307 SEQ ID 8992 GBS308 SEQ ID 8880 GBS309 SEQ ID 3386 GBS310 SEQ ID 286 GBS311 SEQ ID 3964 GBS312 SEQ ID 4660 GBS313 SEQ ID 4090 GBS314 SEQ ID 8556 GBS315 SEQ ID 1766 GBS316 SEQ ID 2000 GBS317 SEQ ID 4210 GBS318 SEQ ID 8548 GBS319 SEQ ID 892 GBS320 SEQ ID 916 GBS321 SEQ ID 8846 GBS322 SEQ ID 8540 GBS323 SEQ ID 2102 GBS324 SEQ ID 8490 GBS325 SEQ ID 8900 GBS326 SEQ ID 8630 GBS327 SEQ ID 5856 GBS328 SEQ ID 6016 GBS329 SEQ ID 8928 GBS330 SEQ ID 8792 GBS331 SEQ ID 922 GBS332 SEQ ID 1004 GBS333 SEQ ID 1786 GBS334 SEQ ID 1784 GBS335 SEQ ID 1782 GBS336 SEQ ID 1886 GBS337 SEQ ID 2010 GBS338 SEQ ID 8638 GBS339 SEQ ID 2080 GBS340 SEQ ID 8594 & 8596 GBS341 SEQ ID 2280 GBS342 SEQ ID 2266 GBS343 SEQ ID 8644 GBS344 SEQ ID 8662 GBS345 SEQ ID 2442 GBS346 SEQ ID 2768 GBS347 SEQ ID 2766 GBS348 SEQ ID 8658 GBS349 SEQ ID 2360 GBS350 SEQ ID 8698 GBS351 SEQ ID 2970 GBS352 SEQ ID 8692 GBS353 SEQ ID 3454 GBS354 SEQ ID 8754 GBS355 SEQ ID 8752 GBS356 SEQ ID 8724 GBS357 SEQ ID 8720 GBS358 SEQ ID 3184 GBS359 SEQ ID 3948 GBS360 SEQ ID 3926 GBS361 SEQ ID 8770 GBS362 SEQ ID 8768 GBS363 SEQ ID 3816 GBS364 SEQ ID 1452 GBS365 SEQ ID 1398 GBS366 SEQ ID 8574 GBS367 SEQ ID 1340 GBS368 SEQ ID 1598 GBS369 SEQ ID 4822 GBS370 SEQ ID 8844 GBS371 SEQ ID 4926 GBS372 SEQ ID 4956 GBS373 SEQ ID 5062 GBS374 SEQ ID 8878 GBS375 SEQ ID 326 GBS376 SEQ ID 5380 GBS377 SEQ ID 5468 GBS378 SEQ ID 5570 GBS379 SEQ ID 8918 GBS380 SEQ ID 156 GBS381 SEQ ID 8934 GBS382 SEQ ID 8610 GBS383 SEQ ID 4738 GBS384 SEQ ID 8836 GBS385 SEQ ID 1094 GBS386 SEQ ID 9038 GBS387 SEQ ID 8558 GBS388 SEQ ID 9040 GBS389 SEQ ID 8516 GBS390 SEQ ID 8952 GBS391 SEQ ID 8522 GBS392 SEQ ID 6220 GBS393 SEQ ID 8966 GBS394 SEQ ID 8960 GBS395 SEQ ID 6276 GBS396 SEQ ID 8468 GBS397 SEQ ID 6262 GBS398 SEQ ID 8806 GBS399 SEQ ID 1960 GBS400 SEQ ID 3154 GBS401 SEQ ID 3170 GBS402 SEQ ID 4236 GBS403 SEQ ID 8798 GBS404 SEQ ID 8800 GBS405 SEQ ID 8508 GBS406 SEQ ID 8506 GBS407 SEQ ID 6484 GBS408 SEQ ID 9042 GBS409 SEQ ID 6678 GBS410 SEQ ID 4064 GBS411 SEQ ID 9044 GBS412 SEQ ID 9046 GBS413 SEQ ID 272 GBS414 SEQ ID 8946 GBS415 SEQ ID 8944 GBS416 SEQ ID 6044 GBS417 SEQ ID 1874 GBS418 SEQ ID 5146 GBS419 SEQ ID 2638 GBS420 SEQ ID 2104 GBS421 SEQ ID 2108 GBS422 SEQ ID 714 GBS423 SEQ ID 6884 GBS424 SEQ ID 4874 GBS425 SEQ ID 3978 GBS426 SEQ ID 3976 GBS427 SEQ ID 6958 GBS428 SEQ ID 3398 GBS429 SEQ ID 3402 GBS430 SEQ ID 8840 GBS431 SEQ ID 8902 GBS432 SEQ ID 8534 GBS433 SEQ ID 2558 GBS434 SEQ ID 8590 GBS435 SEQ ID 484 GBS436 SEQ ID 8472 GBS437 SEQ ID 466 GBS438 SEQ ID 362 GBS439 SEQ ID 900 GBS440 SEQ ID 8536 GBS441 SEQ ID 936 GBS442 SEQ ID 940 GBS443 SEQ ID 998 GBS444 SEQ ID 1776 GBS445 SEQ ID 8634 GBS446 SEQ ID 2048 GBS447 SEQ ID 1654 GBS448 SEQ ID 8592 GBS449 SEQ ID 1634 GBS450 SEQ ID 1630 GBS451 SEQ ID 2098 GBS452 SEQ ID 2062 GBS453 SEQ ID 8636 GBS454 SEQ ID 1734 GBS455 SEQ ID 1690 GBS456 SEQ ID 1684 GBS457 SEQ ID 8656 GBS458 SEQ ID 8650 GBS459 SEQ ID 2152 GBS460 SEQ ID 2148 GBS461 SEQ ID 2394 GBS462 SEQ ID 2778 GBS463 SEQ ID 8688 GBS464 SEQ ID 8684 GBS465 SEQ ID 8682 GBS466 SEQ ID 2694 GBS467 SEQ ID 2350 GBS468 SEQ ID 8660 GBS469 SEQ ID 2998 GBS470 SEQ ID 2988 GBS471 SEQ ID 2924 GBS472 SEQ ID 2910 GBS473 SEQ ID 2882 GBS474 SEQ ID 2878 GBS475 SEQ ID 2856 GBS476 SEQ ID 8690 GBS477 SEQ ID 3112 GBS478 SEQ ID 3432 GBS479 SEQ ID 3460 GBS480 SEQ ID 3504 GBS481 SEQ ID 8734 GBS482 SEQ ID 8740 GBS483 SEQ ID 3606 GBS484 SEQ ID 3562 GBS485 SEQ ID 3552 GBS486 SEQ ID 3762 GBS487 SEQ ID 3756 GBS488 SEQ ID 3732 GBS489 SEQ ID 3730 GBS490 SEQ ID 3704 GBS491 SEQ ID 3698 GBS492 SEQ ID 3252 GBS493 SEQ ID 3244 GBS494 SEQ ID 3238 GBS495 SEQ ID 8722 GBS496 SEQ ID 8716 GBS497 SEQ ID 3876 GBS498 SEQ ID 3858 GBS499 SEQ ID 8758 GBS500 SEQ ID 4022 GBS501 SEQ ID 4106 GBS502 SEQ ID 1406 GBS503 SEQ ID 8580 GBS504 SEQ ID 4578 GBS505 SEQ ID 4566 GBS506 SEQ ID 8832 GBS507 SEQ ID 8830 GBS508 SEQ ID 4644 GBS509 SEQ ID 8828 GBS510 SEQ ID 8826 GBS511 SEQ ID 4892 GBS512 SEQ ID 4970 GBS513 SEQ ID 4974 GBS514 SEQ ID 8862 GBS515 SEQ ID 8864 GBS516 SEQ ID 8866 GBS517 SEQ ID 8868 GBS518 SEQ ID 9012 GBS519 SEQ ID 5068 GBS520 SEQ ID 8870 GBS521 SEQ ID 5228 GBS522 SEQ ID 322 GBS523 SEQ ID 8492 GBS524 SEQ ID 8894 GBS525 SEQ ID 5430 GBS526 SEQ ID 5414 GBS527 SEQ ID 5524 GBS528 SEQ ID 8898 GBS529 SEQ ID 5670 GBS530 SEQ ID 5630 GBS531 SEQ ID 5588 GBS532 SEQ ID 1324 GBS533 SEQ ID 8914 GBS534 SEQ ID 8550 GBS535 SEQ ID 8568 GBS536 SEQ ID 1288 GBS537 SEQ ID 5798 GBS538 SEQ ID 8920 GBS539 SEQ ID 158 GBS540 SEQ ID 8482 GBS541 SEQ ID 184 GBS542 SEQ ID 9048 GBS543 SEQ ID 8932 GBS544 SEQ ID 5880 GBS545 SEQ ID 44 GBS546 SEQ ID 9014 GBS547 SEQ ID 12 GBS548 SEQ ID 8614 GBS549 SEQ ID 8612 GBS550 SEQ ID 4720 GBS551 SEQ ID 4710 GBS552 SEQ ID 1086 GBS553 SEQ ID 1088 GBS554 SEQ ID 1138 GBS555 SEQ ID 8748 GBS556 SEQ ID 5968 GBS557 SEQ ID 774 GBS558 SEQ ID 1192 GBS559 SEQ ID 1196 GBS560 SEQ ID 1268 GBS561 SEQ ID 8518 GBS562 SEQ ID 8676 GBS563 SEQ ID 2296 GBS564 SEQ ID 2300 GBS565 SEQ ID 8950 GBS566 SEQ ID 694 GBS567 SEQ ID 680 GBS568 SEQ ID 6300 GBS569 SEQ ID 8956 GBS570 SEQ ID 8972 GBS571 SEQ ID 8970 GBS572 SEQ ID 3300 GBS573 SEQ ID 3304 GBS574 SEQ ID 8726 GBS575 SEQ ID 8810 GBS576 SEQ ID 4418 GBS577 SEQ ID 8808 GBS578 SEQ ID 4382 GBS579 SEQ ID 4378 GBS580 SEQ ID 1932 GBS581 SEQ ID 8622 GBS582 SEQ ID 8624 GBS583 SEQ ID 1962 GBS584 SEQ ID 8708 GBS585 SEQ ID 8672 GBS586 SEQ ID 6444 GBS587 SEQ ID 8976 GBS588 SEQ ID 8804 GBS589 SEQ ID 8514 GBS590 SEQ ID 8510 GBS591 SEQ ID 630 GBS592 SEQ ID 8504 GBS593 SEQ ID 514 GBS594 SEQ ID 8978 GBS595 SEQ ID 6738 GBS596 SEQ ID 6712 GBS597 SEQ ID 6686 GBS598 SEQ ID 6674 GBS599 SEQ ID 6662 GBS600 SEQ ID 8988 GBS601 SEQ ID 8578 GBS602 SEQ ID 8948 GBS603 SEQ ID 6132 GBS604 SEQ ID 5282 GBS605 SEQ ID 5302 GBS606 SEQ ID 8884 GBS607 SEQ ID 5314 GBS608 SEQ ID 8886 GBS609 SEQ ID 8888 GBS610 SEQ ID 8890 GBS611 SEQ ID 6028 GBS612 SEQ ID 8474 GBS613 SEQ ID 5092 GBS614 SEQ ID 8872 GBS615 SEQ ID 6052 GBS616 SEQ ID 8940 GBS617 SEQ ID 1824 GBS618 SEQ ID 6600 GBS619 SEQ ID 6608 GBS620 SEQ ID 6620 GBS621 SEQ ID 864 GBS622 SEQ ID 8640 GBS623 SEQ ID 8996 GBS624 SEQ ID 9050 GBS625 SEQ ID 2812 GBS626 SEQ ID 8858 GBS627 SEQ ID 8852 GBS628 SEQ ID 8784 GBS629 SEQ ID 6950 GBS630 SEQ ID 4502 GBS631 SEQ ID 4492 GBS632 SEQ ID 4488 GBS633 SEQ ID 8728 GBS634 SEQ ID 3066 GBS635 SEQ ID 8838 GBS636 SEQ ID 4772 GBS637 SEQ ID 8626 GBS638 SEQ ID 8984 GBS639 SEQ ID 8546 GBS640 SEQ ID 6780 GBS641 SEQ ID 900 GBS642 1312 GBS643 1772 GBS644 1956 GBS645 2726 GBS646 3348 GBS647 3770 GBS648 4934 GBS649 5076 GBS650 5446 GBS651 5602 GBS652 5610 GBS653 5760 GBS654 6096 GBS655 6656 GBS656 9324 GBS657 10782 GBS658 8802 GBS659 9344 GBS660 9410 GBS661 9428 GBS662 9286 GBS663 9294 GBS664 9034 GBS665 10546 GBS666 10610 GBS667 9052 GBS668 9036 GBS669 9010 GBS670 10730 GBS671 9020 GBS672 9052 GBS673 9036 GBS674 9034 GBS675 10634 GBS676 10692 GBS677 10746 GBS678 9330 GBS679 9404 GBS680 6668 GBS681 4264 GBS682 6762 GBS683 9290 GBS684 9614 GBS685 10454 GBS686 2774 GBS687 4620 GBS688 10224

TABLE V NUCLEOTIDES DELETED IN EXPRESSION OF GBSnnn PROTEINS GBS Deleted nucleotides  11d 1-153  31d 1-129  64d 1-165  68d 2029-2796   70d 1-402  74d 1-975  79d 1-201 105dN 2689-4119  105dC  1-2688 105d  1-2688 109d 1-120 130d 1-518 170d 1-111 182d 1596-1674  195C  1-1710 195N 1711-3243  209d 757-912  210d 1-99 & 777-879 220d 1-120 231d 1-54  235d 1-270 246d 1-75  248d 1-591 272d 1-531 277d 1-318 281d 1-54  287d 1-108 288d 1-72  293C  1-1229 293N 1230-2379  317N 1729-4107  317C  1-2379 326N 1707-2652  326dN 2326-3927  327N 3034-6831  327C  1-3033 333d 1-150 339d 1-111 352d 1-158 362N 1707-2652  362C  1-1706 397d 1-348 399d 1-111 407d 1174-1473  409d 1-297 424d 1327-1671 

TABLE VI PREDICTED FUNCTIONS FOR CERTAIN SEQ IDs SEQ ID Function 6 manganese ABC transporter, ATP-binding protein (psaB) 12 iron (chelated) ABC transporter, permease protein (psaC) 18 peptidyl-prolyl cis-trans isomerase, cyclophilin-type 26 chorismate binding enzyme (pabB) 30 probable transposase (insertion sequence IS861) 42 peptidase, M20/M25/M40 family 44 drug transporter 50 ribosomal protein L11 (rplK) 54 ribosomal protein L1 (rplA) 62 peptide ABC transporter, permease protein 66 peptide ABC transporter, permease protein 78 uridylate kinase (pyrH) 84 ribosome recycling factor (frr) 104 PhoH family protein (phoH) 110 MutT/nudix family protein superfamily 116 tetracenomycin polyketide synthesis O-methyltransferase TcmP 134 phosphopantetheine adenylyltransferase (coaD) 140 PDZ domain protein 144 5-nucleotidase family protein 156 VanZF-related protein 158 ABC transporter, ATP-binding/permease protein 162 ABC transporter, ATP-binding/permease protein 168 BioY family protein 180 acetyl-CoA acetyltransferase 188 endonuclease III (nth) 196 glucokinase (gki) 200 rhodanese family protein 204 elongation factor Tu family protein (typA) 212 UDP-N-acetylglucosamine--N-acetylmuramyl-(pentapeptide) pyrophosphoryl- 216 cell division protein DivIB 220 cell division protein FtsA (ftsA) 224 cell division protein FtsZ (ftsZ) 236 ylmH protein (ylmH) 240 cell division protein DivIVA (divIVA) 244 isoleucyl-tRNA synthetase (ileS) 252 MutT/nudix family protein 256 ATP-dependent Clp protease, ATP-binding subunit ClpE (clpE) 268 methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cycloh 274 exodeoxyribonuclease VII, large subunit (xseA) 278 exodeoxyribonuclease VII, small subunit (xseB) 282 geranyltranstransferase (ispA) 286 hemolysin A 290 transcriptional repressor 296 DNA repair protein RecN (recN) 300 degV family protein (degV) 322 peptide ABC transporter, permease protein (oppC) 326 peptide ABC transporter, ATP-binding protein (oppD) 328 peptide ABC transporter, ATP-binding protein (oppF) 348 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (ispE) 352 adc operon repressor AdcR (adcR) 356 zinc ABC transporter, ATP-binding protein (adcC) 370 tyrosyl-tRNA synthetase (tyrS) 374 penicillin-binding protein 1B (pbp1B) 378 DNA-directed RNA polymerase, beta subunit (rpoB) 382 dna-directed rna polymerase beta′ chain 390 competence protein CglA (cglA) 406 acetate kinase (ackA) 410 transcriptional regulator 418 pyrroline-5-carboxylate reductase (proC) 422 glutamyl-aminopeptidase (pepA) 432 thioredoxin family protein 436 tRNA binding domain protein (pheT) 440 methyltransferase 442 single-strand DNA-binding protein, authentic point mutation (ssbB) 454 GAF domain protein (lytS) 466 IrgB protein (IrgB) 474 oligopeptide ABC transporter, permease protein 476 peptide ABC transporter, ATP-binding protein 480 peptide ABC transporter, ATP-binding protein (oppF) 484 PTS system, IIABC components (treB) 488 alpha amylase family protein (treC) 494 transcriptional regulator, BglG family 506 transcriptional regulator, BglG family 508 PTS system, IIB component 514 PTS system, IIC component 518 transketolase, N-terminal subunit (tktA) 528 ribosomal protein S15 (rpsO) 546 cysteinyl-tRNA synthetase (cysS) 554 RNA methyltransferase, TrmH family, group 3 562 DegV family protein (degV) 572 ribosomal protein S9 (rpsl) 576 integrase, phage family 580 transcriptional regulator 596 recombination protein 626 transcriptional regulator MutR 630 transporter 640 amino acid ABC transporter, permease protein (opuBB) 642 glycine betaine/L-proline transport ATP binding subunit (proV) 654 lectin, alpha subunit precursor 662 transcriptional regulator 664 acetyltransferase, GNAT family 666 acetyltransferase, GNAT family (rimJ) 670 acetyltransferase, GNAT family 676 transcriptional regulator, tetR family domain protein 680 ABC transporter efflux protein, DrrB family 690 IS1381, transposase OrfA/OrfB, truncation 714 magnesium transporter, CorA family 718 oxidoreductase, Gfo/ldh/MocA family 722 valyl-tRNA synthetase (valS) 730 acetyltransferase, GNAT family 746 methyltransferase 750 bacteriophage L54a, integrase 754 DNA-damage-inducible protein J 774 cation efflux system protein 778 oxidoreductase, aldo/keto reductase family 784 alcohol dehydrogenase, zinc-containing 790 3-oxoadipate enol-lactone hydrolase/4-carboxymuconolactone decarboxylas 804 ribonucleoside-diphosphate reductase, alpha subunit (nrdE) 808 nrdI protein (nrdI) 812 Ribonucleotide reductases 824 elaA protein (elaA) 828 RNA methyltransferase, TrmA family 832 RecX family protein 840 -identity (jag) 844 membrane protein, 60 kDa (yidC) 856 UTP-glucose-1-phosphate uridylyltransferase (galU) 864 rhomboid family protein 884 MORN motif family 892 transcriptional regulator 896 adenylosuccinate lyase (purB) 908 phosphoribosylaminoimidazole carboxylase, catalytic subunit (purE) 912 phosphoribosylamine--glycine ligase (purD) 916 phosphosugar-binding transcriptional regulator 920 acetyl xylan esterase 922 ROK family protein (gki) 926 N-acetylneuraminate lyase (nanA) 936 sugar ABC transporter, permease protein 940 sugar ABC transporter, permease protein (msmF) 952 LysM domain protein, authentic frameshift 956 zoocin A endopeptidase 958 phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydr 962 acetyltransferase, GNAT family family 964 phosphoribosylglycinamide formyltransferase (purN) 968 phosphoribosylformylglycinamidine cyclo-ligase (purM) 972 amidophosphoribosyltransferase (purF) 980 phosphoribosylformylglycinamidine synthase 984 phosphoribosylaminoimidazole-succinocarboxamide synthase (purC) 1042 oligoendopeptidase F (pepF) 1060 ebsC protein 1068 hydrolase, haloacid dehalogenase-like family 1076 riboflavin synthase, beta subunit (ribH) 1082 riboflavin biosynthesis protein RibD (ribD) 1086 Mn2+/Fe2+ transporter, NRAMP family 1094 peptidase, U32 family 1116 HPr(Ser) kinase/phosphatase (hprK) 1130 oxidoreductase 1148 signal recognition particle-docking protein FtsY (ftsY) 1152 Cof family protein 1156 Cof family protein 1172 vicX protein (vicX) 1176 sensory box sensor histidine kinase (vicK) 1180 DNA-binding response regulator (vicR) 1184 amino acid ABC transporter, ATP-binding protein 1188 amino acid ABC transporter, amino acid-binding protein (fliY) 1192 amino acid ABC transporter, permease protein 1196 amino acid ABC transporter, permease protein 1208 DNA-binding response regulator (vicR) 1210 threonyl-tRNA synthetase (thrS) 1214 glycosyl transferase, group 1 1218 glycosyl transferase, group 1 (cpoA) 1222 alpha-amylase (amy) 1230 proline dipeptidase (pepQ) 1238 haloacid dehalogenase-like hydrolase superfamily 1244 mannonate dehydratase (uxuA) 1248 glucuronate isomerase 1254 transcriptional regulator, GntR family 1268 sodiumgalactoside symporter family protein 1270 D-isomer specific 2-hydroxyacid dehydrogenase family protein 1282 transcriptional regulator, LysR family 1290 ABC transporter, ATP-binding protein (potA) 1296 DedA family protein 1308 MutT/nudix family protein family 1310 phosphoserine phosphatase SerB (serB) 1312 septation ring formation regulator EzrA 1320 hydrolase, haloacid dehalogenase-like family (gph) 1340 sensor histidine kinase (vncS) 1348 transmembrane protein Vexp3 (vex3) 1352 ABC transporter, ATP-binding protein (vex2) 1358 transmembrane protein Vexp1 (vex1) 1366 transposase 1374 integrase, phage family 1390 holin 2 1398 minor structural protein 1400 host specificity protein 1404 minor structural protein 1406 PblA 1486 homeobox protein drg11 1488 reverse transcriptase 1496 p22 erf-like protein 1498 gp157 1500 tropomyosin 2 1512 gp49 homologous 1526 transcriptional regulator-related protein 1566 chorismate mutase 1572 PTS system component 1576 PTS system, IIB component 1580 PTS system IIA component 1584 lactose phosphotransferase system repressor (lacR) 1594 adhesion lipoprotein (lmb) 1602 GTP pyrophosphokinase (relA) 1606 2′,3′-cyclic-nucleotide 2′-phosphodiesterase (cpdB) 1616 iron ABC transporter, iron-binding protein 1620 DNA-binding response regulator 1630 PTS system component 1634 PTS system component (manM) 1638 PTS system component (manL) 1642 PTS system component 1658 response regulator BlpR (blpR) 1676 phosphate transport system regulatory protein PhoU 1680 phosphate ABC transporter, ATP-binding protein (pstB) 1684 phosphate ABC transporter, permease protein (pstA) 1690 phosphate ABC transporter, permease protein (pstC) 1694 probable hemolysin precursor 1704 ribosomal protein L11 methyltransferase (prmA) 1710 transcriptional regulator, MerR family (skgA) 1714 acetyltransferase, GNAT family 1716 MutT/nudix family protein 1722 spermidine N1-acetyltransferase 1726 ATPase, AAA family 1736 ABC transporter domain protein 1738 Helix-turn-helix domain protein 1748 integrase, phage family 1756 Helix-turn-helix domain protein 1762 bacteriophage L54a, integrase 1768 LPXTG-motif cell wall anchor domain protein 1776 membrane protein 1778 conjugal transfer protein 1780 IS1381, transposase OrfA/OrfB, truncation 1802 transcriptional regulator (rstR-1) 1806 transcriptional regulator 1808 FtsK/SpoIIIE family protein 1814 aggregation substance 1818 mercuric reductase 1822 transcriptional regulator, MerR family 1824 Mn2+/Fe2+ transporter, NRAMP family 1830 ABC transporter, ATP-binding protein (epiF) 1848 Helix-turn-helix domain protein 1850 type 2 phosphatidic acid phosphatase(PAP2), family 1858 Abortive infection protein family 1868 aminotransferase, class-V 1874 glutathione reductase (gor) 1882 chorismate synthase (aroC) 1886 3-dehydroquinate synthase (aroB) 1900 sulfatase family protein 1914 ABC transporter, ATP-binding protein 1920 smf protein (Smffamily) 1924 transferrin receptor 1928 iron compound ABC transporter, ATP-binding protein 1932 iron compound ABC transporter, permease protein 1942 acetyltransferase, CysE/LacA/LpxA/NodL family 1952 GTP-binding protein 1958 carbon starvation protein A 1960 response regulator (lytR) 1962 GAF domain protein (lytS) 2000 extracellular protein 2004 diarrheal toxin (yukA) 2024 carbamoyl-phosphate synthase, large subunit (carB) 2028 carbamoyl-phosphate synthase, small subunit (carA) 2032 aspartate carbamoyltransferase (pyrB) 2036 dihydroorotase, multifunctional complex type (pyrC) 2040 orotate phosphoribosyltransferase (pyrE) 2048 membrane protein 2062 phosphate ABC transporter, permease protein (pstA-2) 2064 phosphate ABC transporter, ATP-binding protein (pstB) 2070 phosphate transport system regulatory protein PhoU 2072 aminopeptidase N (pepN) 2076 DNA-binding response regulator (arlR) 2080 sensor histidine kinase (arlS) 2088 signal recognition particle protein (ffh) 2102 peptide ABC transporter, peptide-binding protein 2104 integrase/recombinase, phage integrase family 2108 sensor histidine kinase 2112 DNA-binding response regulator (vicR) 2118 ABC transporter, ATP-binding protein 2122 nisin-resistance protein 2130 lipoprotein 2136 gid protein (gid) 2140 transcriptional regulator, GntR family 2142 GMP synthase (guaA) 2152 branched-chain amino acid ABC transporter, permease protein (livM) 2154 branched-chain amino acid ABC transporter, ATP-binding protein (livG) 2156 branched-chain amino acid ABC transporter, ATP-binding protein (livF) 2160 acetoin utilization protein AcuB 2174 DNA polymerase III, delta prime subunit (holB) 2186 copper homeostasis protein (cutC) 2190 phosphoserine aminotransferase (serC) 2202 methylated-DNA--protein-cysteine S-methyltransferase (ogt) 2208 exodeoxyribonuclease III (xth) 2214 PTS system, IIC component 2224 tellurite resistance protein TehB (tehB) 2246 icaA protein 2250 acetyltransferase, GNAT family 2258 oxidoreductase, short chain dehydrogenase/reductase family (fabG) 2266 oxidoreductase, Gfo/Idh/MocA family family 2268 glyoxalase family protein 2272 UDP-N-acetylglucosamine pyrophosphorylase (glmU) 2276 MutT/nudix family protein 2284 5-methylthioadenosine/S-adenosylhomocysteine nucleosidase (mtf) 2296 phosphatidate cytidylyltransferase (cdsA) 2300 membrane-associated zinc metalloprotease 2308 autolysin (flgJ) 2312 DNA polymerase III, alpha subunit, Gram-positive type 2320 nitroreductase family protein superfamily 2326 4-hydroxy-2-oxoglutarate aldolase/2-deydro-3-deoxyphosphogluconate aldo 2328 carbohydrate kinase, PfkB family 2336 oxidoreductase, short chain dehydrogenase/reductase family (fabG) 2338 PTS system, IIA component (manL) 2342 glucuronyl hydrolase 2346 PTS system, IIB component (manL) 2350 PTS system, IIC component (manM) 2364 sugar binding transcriptional regulator RegR (regR) 2368 polypeptide deformylase (def) 2380 oxidoreductase, Gfo/Idh/MocA family 2382 endopeptidase O (pepO) 2394 Na+/H+ antiporter 2404 transcriptional regulator 2410 replication initiation protein RepRC 2412 bacteriophage L54a, antirepressor 2416 e11 2422 replicative DNA helicase (dnaB) 2432 GTP-binding protein 2440 arpR protein 2444 gene 17 protein 2458 integrase/recombinase, phage integrase family 2468 bacteriophage L54a, phage D3 terminase 2472 protease 2500 PblB 2504 sensor histidine kinase 2514 N-acetylmuramoyl-L-alanine amidase 2518 KH domain protein 2522 ribosomal protein S16 (rpsP) 2526 permease 2528 ABC transporter, ATP-binding protein 2538 carbamoyl-phosphate synthase, large subunit 2540 carbamoyl-phosphate synthase, small subunit (carA) 2550 transcriptional regulator, LysR family 2554 ribosomal protein L27 (rpmA) 2562 ribosomal protein L21 (rplU) 2572 glycerophosphoryl diester phosphodiesterase 2582 nitroreductase family protein 2586 dipeptidase (pepV) 2614 GTP-binding protein HflX (hflX) 2618 galactose-1-phosphate uridylyltransferase (galT) 2626 oxidoreductase, short chain dehydrogenase/reductase family 2630 single-stranded-DNA-specific exonuclease RecJ (recJ) 2638 adenine phosphoribosyltransferase (apt) 2646 Bcl-2 family protein 2654 oxidoreductase, DadA family protein 2658 glucose-1-phosphate thymidylyltransferase (rfbA) 2664 dTDP-4-dehydrorhamnose 3,5-epimerase (rfbC) 2682 hyaluronidase 2686 mutator MutT protein (mutX) 2690 MutT/nudix family protein 2694 membrane protein 2702 acetolactate synthase (ilvK) 2706 adherence and virulence protein A (pavA) 2714 ABC transporter, permease protein (rbsC) 2722 metallo-beta-lactamase superfamily protein 2734 ribose 5-phosphate isomerase (rpiA) 2738 phosphopentomutase (deoB) 2742 purine nucleoside phosphorylase, family 2 (deoD) 2750 purine nucleoside phosphorylase (deoD) 2762 capsular polysaccharide biosynthesis protein Cps4A (cps4A) 2768 cpsb protein 2770 cpsc protein 2772 CpsE 2774 CpsF 2776 CpsVG 2778 CpsVH 2780 CpsVM 2782 CpsVN 2784 glycosyl transferase domain protein 2786 glycosyl transferase, family 2/glycosyl transferase family 8 2790 CpsVK 2794 CpsL 2796 neuB protein 2798 UDP-N-acetylglucosamine 2-epimerase 2800 hexapeptide transferase family protein 2802 NeuA 2808 uracil-DNA glycosylase (ung) 2818 DNA topoisomerase IV, B subunit (parE) 2822 DNA topoisomerase IV, A subunit (parC) 2826 branched-chain amino acid aminotransferase (ilvE) 2842 glycerol kinase (glpK) 2848 aerobic glycerol-3-phosphate dehydrogenase (glpD) 2874 ABC transporter, ATP-binding protein 2882 PTS system component (bglP) 2886 glutamate 5-kinase (proB) 2890 gamma-glutamyl phosphate reductase (proA) 2898 cell division protein FtsL (ftsL) 2904 penicillin-binding protein 2X (pbpX) 2910 phospho-N-acetylmuramoyl-pentapeptide-transferase (mraY) 2914 ATP-dependent RNA helicase, DEAD/DEAH box family (deaD) 2918 ABC transporter, substrate-binding protein 2924 amino acid ABC transporter, permease protein 2928 amino acid ABC transporter, ATP-binding protein 2932 thioredoxin reductase (trxB) 2940 NAD+ synthetase (nadE) 2944 aminopeptidase C (pepC) 2952 recombination protein U (recU) 2966 Uncharacterized protein family UPF0020 family 2974 autoinducer-2 production protein LuxS (luxS) 2978 KH domain protein 2986 ABC transporter, ATP-binding protein 2994 DNA-binding response regulator (vraR) 3000 guanylate kinase (gmk) 3004 DNA-directed RNA polymerase, omega subunit 3008 primosomal protein N (priA) 3012 methionyl-tRNA formyltransferase (fmt) 3016 Sun protein (sun) 3020 protein phosphatase 2C 3032 sensor histidine kinase 3034 DNA-binding response regulator (vraR) 3036 cof family protein/peptidyl-prolyl cis-trans isomerase, cyclophilin typ 3040 S1 RNA binding domain protein (rpsA) 3044 pyruvate formate-lyase-activating enzyme 3062 PTS system, IIB component (celA) 3066 PTS system, cellobiose-specific IIC component (celB) 3068 formate acetyltransferase (pfl) 3072 transaldolase 3080 cysteine synthase A (cysK) 3088 comF operon protein 1 (comFA) 3092 competence protein ComF 3096 ribosomal subunit interface protein (yfiA) 3104 tryptophanyl-tRNA synthetase (trpS) 3108 carbamate kinase (arcC) 3116 ornithine carbamoyltransferase (argF) 3124 arginine deiminase (arcA) 3134 transcriptional regulator, Crp/Fnr family 3138 inosine-5′-monophosphate dehydrogenase (guaB) 3140 MutR 3142 transporter 3146 recF protein (recF) 3158 peptidase, M16 family 3166 ABC transporter, ATP-binding protein 3170 ABC transporter, ATP-binding protein 3178 LysM domain protein (lytN) 3180 immunodominant antigen A (isaA) 3184 L-serine dehydratase, iron-sulfur-dependent, alpha subunit (sdhA) 3188 L-serine dehydratase, iron-sulfur-dependent, beta subunit (sdhB) 3202 DHH subfamily 1 protein 3206 ribosomal protein L9 (rplI) 3210 replicative DNA helicase (dnaB) 3216 ribosomal protein S4 (rpsD) 3224 transcriptional regulator, TetR family 3236 membrane protein 3238 choline transporter (proWX) 3240 glycine betaine/L-proline transport ATP binding subunit (proV) 3242 DNA-binding response regulator 3244 Histidine kinase-, DNA gyrase B-, phytochrome-like ATPase family 3246 ornithine carbamoyltransferase (argF) 3248 carbamate kinase (arcC) 3252 membrane protein 3256 sensory box histidine kinase VicK 3258 DNA-binding response regulator 3268 Helix-turn-helix domain protein 3278 integrase 3284 ribosomal protein L33 (rpmG) 3288 ribosomal protein L32 (rpmF) 3300 YitT family protein 3304 YitT family protein 3320 DNA mismatch repair protein MutS (mutS) 3324 cold-shock domain family protein-related protein 3336 drug transporter 3340 Holliday junction DNA helicase RuvA (ruvA) 3352 recA protein (recA) 3386 oxidoreductase, Gfo/Idh/MocA family 3390 acetyltransferase, GNAT family 3394 anaerobic ribonucleoside-triphosphate reductase activating protein (nrd 3412 ABC transporter, permease protein (rbsC) 3414 ABC transporter, ATP-binding protein (nrtC) 3416 PTS system, mannose-specific IIAB components (manL) 3420 Cof family protein 3432 xanthine/uracil permease family protein 3440 acetyltransferase, GNAT family 3442 transcriptional regulator (cps4A) 3448 HIT family protein (hit) 3460 ABC transporter, permease protein 3472 Uncharacterized BCR, YhbC family COG0779 superfamily 3484 ribosomal protein L7A family 3496 esterase 3500 transcriptional repressor, CopY (copY) 3504 cation-transporting ATPase, E1-E2 family 3508 cation-binding protein-related protein 3520 DNA polymerase I (polA) 3534 DNA-binding response regulator (saeR) 3536 sensor histidine kinase (saeS) 3562 drug resistance transporter, EmrB/QacA subfamily 3566 peptidase M24 family protein 3570 peptidase M24 family protein (pepQ) 3572 cytidine/deoxycytidylate deaminase family protein 3584 translation elongation factor P (efp) 3592 N utilization substance protein B (nusB) 3596 sugar-binding transcriptional regulator, LacI family (scrR) 3600 sucrose-6-phosphate dehydrogenase (scrB) 3606 PTS system IIABC components (scrA) 3610 fructokinase (scrK) 3614 mannose-6-phosphate isomerase, class I (manA) 3622 phospho-2-dehydro-3-deoxyheptonate aldolase (aroH) 3626 holo-(acyl-carrier-protein) synthase (acpS) 3630 alanine racemase (alr) 3634 autolysin (usp45) 3636 ATP-dependent DNA helicase RecG (recG) 3642 shikimate 5-dehydrogenase (aroE) 3652 Cof family protein 3668 ferredoxin-related protein 3676 peptidase t (pepT) 3684 UDP-N-acetylmuramoylalanyl-D-glutamate--2,6-diaminopimelate ligase (mur 3692 iron compound ABC transporter, substrate-binding protein 3698 FecCD transport family protein (sirB) 3704 iron compound ABC transporter, permease protein (sirB) 3710 inorganic pyrophosphatase, manganese-dependent (ppaC) 3714 pyruvate formate-lyase-activating enzyme (pflA) 3718 CBS domain protein 3730 acid phosphatase 3736 LPXTG-motif cell wall anchor domain protein 3738 LPXTG-site transpeptidase family protein 3742 LPXTG-site transpeptidase family protein 3744 cell wall surface anchor family protein 3746 cell wall surface anchor family protein 3752 glycosyl transferase, group 1 family protein domain protein 3754 EpsQ protein 3756 polysaccharide extrusion protein 3768 dTDP-glucose 4-6-dehydratase 3782 glycosyl transferase domain protein 3788 dTDP-4-dehydrorhamnose reductase (rfbD) 3796 RNA polymerase sigma-70 factor (rpoD) 3802 DNA primase (dnaG) 3816 ABC transporter, ATP-binding protein Vexp2 (vex2) 3818 permease 3820 transmembrane protein Vexp3 3822 transmembrane protein Vexp3 3832 endopeptidase O (pepO) 3834 endopeptidase O (pepO) 3840 serine protease, subtilase family 3842 exotoxin 2 3844 CylK 3854 glycine cleavage system T protein 3856 CylE 3858 ABC transporter homolog CylB 3862 acyl carrier protein homolog AcpC (acpP) 3864 3-oxoacyl-(acyl-carrier-protein) reductase (fabG) 3868 CylD 3876 membrane protein 3912 LPXTG-site transpeptidase family protein 3916 LPXTG-site transpeptidase family protein 3918 LPXTG-site transpeptidase family protein 3920 LPXTG-motif cell wall anchor domain protein 3928 chaperonin, 33 kDa (hslO) 3932 Tn5252, Orf 10 protein 3934 transposase OrfAB, subunit B 3948 psr protein 3952 shikimate kinase (aroK) 3964 enolase (eno) 3972 MutT/nudix family protein 3976 glycosyl transferase, group 1 3978 preprotein translocase, SecA subunit (secA) 3986 preprotein translocase SecY family protein 3990 glycosyl transferase, family 8 3992 glycosyl transferase, family 2 3998 glycosyl transferase, family 8 4000 glycosyl transferase, family 2/glycosyl transferase family 8 4002 glycosyl transferase, family 8 4012 LPXTG-motif cell wall anchor domain protein (clfB) 4016 transcriptional regulator 4018 excinuclease ABC, B subunit (uvrB) 4022 Abortive infection protein family 4024 amino acid ABC transporter, amino acid-binding protein/permease protein 4026 amino acid ABC transporter, ATP-binding protein 4034 GTP-binding protein, GTP1/Obg family (obg) 4042 aminopeptidase PepS (pepS) 4050 ribosomal small subunit pseudouridine synthase A (rsuA) 4060 lactoylglutathione lyase (gloA) 4064 glycosyl transferase family protein 4072 alkylphosphonate utilization operon protein PhnA (phnA) 4078 glucosamine--fructose-6-phosphate aminotransferase (isomerizing) (glmS) 4090 Phosphofructokinase 4094 DNA polymerase III, alpha subunit (dnaE) 4098 transcriptional regulator, GntR family 4102 ABC transporter, ATP-binding protein 4106 ABC transporter, ATP-binding protein 4116 FtsK/SpoIIIE family protein 4122 Helix-turn-helix domain protein 4152 Helix-turn-helix domain protein 4158 excisionase 4160 transposase 4166 chloramphenicol acetyltransferase (cat) 4174 PilB-related protein 4178 acetyltransferase 4182 Leucine Rich Repeat domain protein 4190 nucleoside diphosphate kinase (ndk) 4206 Protein of unknown function superfamily 4218 hydrolase, haloacid dehalogenase-like family (pho2) 4226 oxygen-independent coproporphyrinogen III oxidase 4236 phosphoglucomutase/phosphomannomutase family protein (femD) 4240 Gram-positive signal peptide, YSIRK family domain protein 4256 cobyric acid synthase (cobQ) 4260 lipoate-protein ligase A (lplA) 4264 branched-chain alpha-keto acid dehydrogenase E3 component, lipoamide de 4266 pyruvate dehydrogenase complex, E2 component, dihydrolipoamide acetyltr 4270 pyruvate dehydrogenase complex, E1 component, pyruvate dehydrogenase be 4286 magnesium transporter, CorA family 4294 exonuclease RexB (rexB) 4302 phenylalanyl-tRNA synthetase, beta subunit (pheT) 4324 ATP synthase F1, epsilon subunit (atpC) 4328 ATP synthase F1, beta subunit (atpD) 4332 ATP synthase F1, gamma subunit (atpG) 4338 ATP synthase F1, alpha subunit (atpA) 4342 ATP synthase F1, delta subunit (atpH) 4346 ATP synthase F0, B subunit (atpF) 4350 ATP synthase, F0 subunit A (atpB) 4354 proton-translocating ATPase, c subunit-related protein 4360 glycogen synthase (glgA) 4362 glycogen biosynthesis protein GlgD (glgD) 4366 1,4-alpha-glucan branching enzyme (glgB) 4368 pullulanase 4382 ribonuclease BN 4396 acetyltransferase, GNAT family 4398 UDP-N-acetylglucosamine 1-carboxyvinyltransferase (murA) 4402 thiamine-phosphate pyrophosphorylase (thiE) 4406 phosphomethylpyrimidine kinase (thiD) 4410 transcriptional regulator, Deg family (tenA) 4414 ABC transporter, ATP-binding protein 4426 S-adenosylmethionine synthetase (metK) 4440 DNA polymerase III, gamma and tau subunits (dnaX) 4444 GAF domain protein 4448 uridine kinase (udk) 4452 ATP-dependent RNA helicase, DEAD/DEAH box family 4458 peptidoglycan GlcNAc deacetylase (pgdA) 4462 glyceraldehyde-3-phosphate dehydrogenase, NADP-dependent (gapN) 4466 phosphoenolpyruvate-protein phosphotransferase (ptsI) 4470 phosphocarrier protein hpr 4474 NrdH-redoxin-related protein 4478 ribonucleoside-diphosphate reductase 2, alpha subunit (nrdE) 4498 glycosyl transferase, family 8 4504 alanyl-tRNA synthetase (alaS) 4512 alkyl hydroperoxide reductase, subunit F (ahpF) 4516 alkyl hydroperoxide reductase, subunit C (ahpC) 4520 ribosomal protein S2 (rpsB) 4524 translation elongation factor Ts (tsf) 4532 transcriptional regulator CtsR (ctsR) 4536 ATP-dependent Clp protease, ATP-binding subunit (clpC) 4540 deoxynucleoside kinase 4544 NifR3/Smm1 family protein 4548 chaperonin, 33 kDa (hslO) 4558 glutamate--cysteine ligase (gshA) 4562 Helix-turn-helix domain, fis-type protein 4566 perfringolysin O regulator protein (pfoR) 4570 adenylosuccinate synthetase (purA) 4578 SgaT protein (sgaT) 4582 PTS system, IIB component (sgaT) 4586 PTS system, IIA component (mtlA) 4590 hexulose-6-phosphate synthase 4594 hexulose-6-phosphate isomerase 4598 L-ribulose-5-phosphate 4-epimerase (araD) 4606 sugar binding transcriptional regulator RegR 4610 D-isomer specific 2-hydroxyacid dehydrogenase family protein (serA) 4622 transcriptional regulator, BglG family 4632 glycine betaine/L-proline transport ATP binding subunit (proV) 4636 amino acid ABC transporter, permease protein 4644 Na+/H+ exchanger family protein (kefB) 4648 glyoxylase family protein 4652 LPXTG-site transpeptidase family protein 4656 DNA gyrase, A subunit (gyrA) 4660 L-lactate dehydrogenase (ldh) 4664 NADH oxidase (nox) 4680 lipoprotein (bmpD) 4690 pantothenate kinase (coaA) 4694 ribosomal protein S20 (rpsT) 4698 amino acid ABC transporter, amino acid-binding protein (aatB) 4702 amino acid ABC transporter, ATP-binding protein 4726 ribosomal large subunit pseudouridine synthase B (rluB) 4734 Uncharacterized ACR, COG1354 4738 integrase/recombinase, phage integrase family (xerD) 4742 CBS domain protein 4746 phosphoesterase 4750 HAM1 protein 4768 transcriptional regulator, biotin repressor family 4792 amino acid ABC transproter, permease protein 4796 amino acid ABC transporter, substrate-binding protein 4798 6-aminohexanoate-cyclic-dimer hydrolase 4800 transcription elongation factor GreA (greA) 4804 Uncharacterized BCR, YceG family COG1559 4812 UDP-N-acetylmuramate--alanine ligase (murC) 4822 Snf2 family protein 4828 GTP-binding protein (b2511) 4832 primosomal protein Dnal (dnal) 4844 sensor histidine kinase (arlS) 4846 DNA-binding response regulator (arlR) 4852 heat shock protein HtpX (htpX) 4870 potassium uptake protein, Trk family 4874 ABC transporter, ATP-binding protein 4888 phosphoglycerate kinase (pgk) 4896 transcriptional regulator, MerR family 4900 glutamine synthetase, type I (glnA) 4904 secreted 45 kd protein (usp45) 4908 metallo-beta-lactamase superfamily protein 4916 glycoprotease family protein 4926 glycoprotease family protein (gcp) 4938 ribosomal protein S14p/S29e (rpsN) 4952 exonuclease (dnaQ) 4956 transcriptional regulator, merR family 4958 cyclopropane-fatty-acyl-phospholipid synthase (cfa) 4970 1,4-dihydroxy-2-naphthoate octaprenyltransferase (menA) 4972 pyridine nucleotide-disulphide oxidoreductase (ndh) 4974 cytochrome d oxidase, subunit I (cydA) 4976 cytochrome d ubiquinol oxidase, subunit II (cydB) 4980 transport ATP-binding protein CydD 4988 polyprenyl synthetase (ispB) 4990 X-pro dipeptidyl-peptidase (pepX) 4998 drug transporter 5002 universal stress protein family 5004 glycerol uptake facilitator protein (glpF) 5012 cppA protein (cppA) 5034 exodeoxyribonuclease V, alpha subunit (recD) 5038 Signal peptidase I 5042 ribonuclease HIII (rnhC) 5062 transcriptional regulator 5068 maltose ABC transporter, permease protein (malD) 5072 maltose ABC transporter, permease protein (malC) 5088 ABC transporter, ATP-binding protein 5092 ABC transporter, permease protein 5106 spspoJ protein (spo0J) 5114 DNA polymerase III, beta subunit (dnaN) 5118 Diacylglycerol kinase catalytic domain (presumed) protein 5138 transcription-repair coupling factor (mfd) 5142 S4 domain protein 5156 MesJ/Ycf62 family protein 5160 hypoxanthine phosphoribosyltransferase (hpt) 5164 cell division protein FtsH (ftsH) 5172 hydrolase, haloacid dehalogenase-like family (b2690) 5178 transcriptional regulator, MarR family 5182 3-oxoacyl-(acyl-carrier-protein) synthase III (fabH) 5190 enoyl-(acyl-carrier-protein) reductase (fabK) 5194 malonyl CoA-acyl carrier protein transacylase (fabD) 5198 3-oxoacyl-[acyl-carrier protein] reductase (fabG) 5200 3-oxoacyl-(acyl-carrier-protein) synthase II (fabF) 5202 acetyl-CoA carboxylase, biotin carboxyl carrier protein (accB) 5206 (3R)-hydroxymyristoyl-(acyl-carrier-protein) dehydratase (fabZ) 5210 acetyl-CoA carboxylase, biotin carboxylase (accC) 5214 acetyl-CoA carboxylase, carboxyl transferase, beta subunit (accD) 5218 acetyl-CoA carboxylase, carboxyl transferase, alpha subunit (accA) 5224 seryl-tRNA synthetase (serS) 5234 PTS system, mannose-specific IID component 5246 ribosomal large subunit pseudouridine synthase, RluD subfamily (rluD) 5254 GTP pyrophosphokinase (relA) 5266 ribose-phosphate pyrophosphokinase (prsA) 5270 aminotransferase, class-V 5274 DNA-binding protein 5282 Domain of unknown function 5290 platelet activating factor 5296 transcriptional regulator, AraC family 5302 voltage-gated chloride channel family protein 5318 spermidine/putrescine ABC transporter, ATP-binding protein (potA) 5320 UDP-N-acetylenolpyruvoylglucosamine reductase (murB) 5324 bifunctional folate synthesis protein (folK) 5328 dihydroneopterin aldolase (folB) 5332 dihydropteroate synthase (folP) 5336 GTP cyclohydrolase I (folE) 5344 rarD protein (rarD) 5348 homoserine kinase (thrB) 5354 Polysaccharide deacetylase family (icaB) 5362 osmoprotectant transporter, BCCT family (opuD) 5384 thiol peroxidase (psaD) 5388 hydrolase 5390 transcriptional regulator, GntR family 5402 gls24 protein 5424 uncharacterized domain 1 5440 cation efflux family protein 5454 dihydroorotate dehydrogenase A (pyrDa) 5458 beta-lactam resistance factor (fibB) 5462 beta-lactam resistance factor (fibA) 5474 HD domain protein 5482 cation-transporting ATPase, E1-E2 family 5486 fructose-1,6-bisphosphatase (fbp) 5488 iron-sulfur cluster-binding protein 5492 peptide chain release factor 2 (prfB) 5496 cell division ABC transporter, ATP-binding protein FtsE (ftsE) 5504 carboxymethylenebutenolidase-related protein 5506 metallo-beta-lactamase superfamily protein 5514 DNA polymerase III, epsilon subunit/ATP-dependent helicase DinG 5520 asparaginyl-tRNA synthetase (asnS) 5526 inosine-uridine preferring nucleoside hydrolase (iunH) 5528 general stress protein 170 5534 Uncharacterised protein family superfamily 5538 Uncharacterized BCR, COG1481 5546 zinc ABC transporter, zinc-binding adhesion liprotein (adcA) 5560 isochorismatase family protein (entB) 5566 3-hydroxybutyryl-CoA dehydrogenase 5572 pyruvate phosphate dikinase (ppdK) 5574 glutamyl-tRNA(Gln) amidotransferase, C subunit (gatC) 5580 glutamyl-tRNA(Gln) amidotransferase, A subunit (gatA) 5594 GTP-binding protein 5612 iojap-related protein 5626 transcriptional regulator SkgA (skgA) 5630 glycerol uptake facilitator protein (glpF) 5634 dihydroxyacetone kinase family protein 5638 dihydroxyacetone kinase family protein 5640 transcriptional regulator, tetR family 5646 dihydroxyacetone kinase family protein 5654 glutamine amidotransferase, class I 5666 peptidase, M20/M25/M40 family 5668 ABC transporter, ATP-binding protein 5686 pur operon repressor (purR) 5690 cmp-binding-factor 1 (cbf1) 5694 competence-induced protein Ccs50 (ccs50) 5702 ribulose-phosphate 3-epimerase (rpe) 5710 rRNA (guanine-N1-)-methyltransferase (rrmA) 5712 dimethyladenosine transferase (ksgA) 5718 primase-related protein 5726 endosome-associated protein 5728 CG17785 gene product 5734 dltD protein (dltD) 5738 D-alanyl carrier protein-related protein 5742 dltB protein (dltB) 5754 DNA-binding response regulator (arlR) 5756 ribosomal protein L34 (rpmH) 5766 penicillin-binding protein 4 (pbp4) 5770 intein-containing protein 5774 NifU family protein 5778 aminotransferase, class-V 5782 Uncharacterized protein family (UPF0051) family 5786 ABC transporter, ATP-binding protein 5790 glycosyl transferase domain protein (llm) 5794 transcriptional regulator MecA (mecA) 5798 undecaprenol kinase 5806 amino acid ABC transporter, amino acid-binding protein/permease protein 5808 amino acid ABC transporter, ATP-binding protein 5834 riboflavin biosynthesis protein RibF (ribF) 5850 type I restriction-modification system, S subunit 5860 lipoprotein 5862 aggregation substance 5866 ID479 5896 type II DNA modification methyltransferase Spn5252IP (spn5252IMP) 5916 ribosomal protein L10 (rplJ) 5922 ATP-dependent Clp protease, ATP-binding subunit ClpC (clpC) 5926 homocysteine S-methyltransferase (mmuM) 5932 transcriptional regulator, TetR family 5938 GTP-binding protein (cgpA) 5952 thymidylate synthase (thyA) 5956 condensing enzyme, FabH-related 5960 hydroxymethylglutaryl-CoA reductase, degradative 5974 gene_idK21C13.21~pir||T04769~strong similarity to unknown protein, put 5976 FMN-dependent dehydrogenase family protein 5980 phosphomevalonate kinase 5986 diphosphomevalonate decarboxylase (mvaD) 5990 mevalonate kinase (mvk) 5994 Histidine kinase-, DNA gyrase B-, phytochrome-like ATPase family (PhoR1 6002 GTP pyrophosphokinase (relA) 6006 transposase for insertion sequence element is904 6016 5′-nucleotidase family 6018 polypeptide deformylase (def) 6022 NADP-specific glutamate dehydrogenase (gdhA) 6026 ABC transporter, ATP-binding/permease protein 6028 ABC transporter, ATP-binding/permease protein 6030 acetyltransferase, GNAT family family 6032 ABC transporter, ATP-binding protein 6040 degV family protein (degV) 6056 carbohydrate kinase, PfkB family (fruB) 6064 beta-lactam resistance factor (fibB) 6070 2-dehydropantoate 2-reductase 6076 PTS system component 6078 pyridine nucleotide-disulphide oxidoreductase family protein (trxB) 6082 tRNA (guanine-N1)-methyltransferase (trmD) 6092 c5a peptidase precursor 6100 ParA 6102 transposase family protein (orfA) 6116 Tn5252, relaxase 6120 Tn5252, Orf 10 protein 6124 mercuric reductase 6126 transcriptional regulator, MerR family 6132 cation transport ATPase, E1-E2 family 6138 cation-transporting ATPase, E1-E2 family 6140 cation-transporting ATPase, E1-E2 family 6144 cation-transporting ATPase, E1-E2 family 6146 transcriptional repressor, CopY (copY) 6150 cadmium resistance transporter 6158 membrane protein 6162 flavoprotein (dfp) 6170 lipoate-protein ligase A 6174 FMN oxidoreductase (nemA) 6178 Bacterial luciferase superfamily 6182 glycine cleavage system H protein (gcvH) 6186 Domain of unknown function 6194 lipoate-protein ligase A (lplA) 6198 formate--tetrahydrofolate ligase (fhs) 6202 cardiolipin synthetase (cls) 6220 aminotransferase, class II (aspB) 6222 RNA methyltransferase, TrmH family, group 2 6232 60 kda chaperonin 6242 purine nucleoside phosphorylase (deoD) 6248 deoxyribose-phosphate aldolase (deoC) 6254 Lyme disease proteins of unknown function 6258 ribosomal large subunit pseudouridine synthase, RluD subfamily (rluD) 6262 penicillin-binding protein 2A (pbp2A) 6266 pathenogenicity protein 6268 transcription antitermination protein NusG (nusG) 6272 glycosyl transferase, family 8 6276 glycosyl transferase, family 8 6284 sugar transporter family protein 6292 sensory box histidine kinase 6306 homocysteine S-methyltransferase (metH) 6310 glycerol dehydrogenase 6312 DNA topology modulation protein FlaR 6316 translation initiation factor IF-1 (infA) 6320 adenylate kinase (adk) 6326 ribosomal protein L15 (rplO) 6330 ribosomal protein L30 (rpmD) 6336 ribosomal protein S5 (rpsE) 6344 ribosomal protein L6 (rplF) 6348 ribosomal protein S8 (rpsH) 6352 ribosomal protein S14 (rpsN) 6356 ribosomal protein L5 (rplE) 6360 ribosomal protein L24 (rplX) 6366 ribosomal protein L14 (rplN) 6368 ribosomal protein S17 (rpsQ) 6372 ribosomal protein L29 (rpmC) 6374 ribosomal protein L16 (rplP) 6378 ribosomal protein S3 (rpsC) 6382 ribosomal protein L22 (rplV) 6386 ribosomal protein S19 (rpsS) 6390 ribosomal protein L2 (rplB) 6394 ribosomal protein L23 (rplW) 6398 ribosomal protein L4/L1 family (rplD) 6402 ribosomal protein L3 (rplC) 6408 ribosomal protein S10 (rpsJ) 6414 MATE efflux family protein 6418 threonine synthase (thrC) 6428 Uncharacterized BCR, COG1636 superfamily 6436 4-alpha-glucanotransferase (malQ) 6440 glycogen phosphorylase family protein (malP) 6444 glycerol-3-phosphate transporter (glpT) 6452 rhodanese family protein 6458 ammonium transporter 6464 DNA repair protein RadA (radA) 6472 oxidoreductase, pyridine nucleotide-disulfide, class I 6478 ribose ABC transporter, periplasmic D-ribose-binding protein (rbsB) 6484 ribose ABC transporter, ATP-binding protein (rbsA) 6486 ribose ABC transporter protein (rbsD) 6488 ribokinase (rbsK) 6498 ABC transporter, ATP-binding protein 6502 DNA-binding response regulator (vicR) 6506 argininosuccinate synthase (argG) 6508 argininosuccinate lyase (argH) 6514 bacteriophage L54a, repressor protein 6528 soluble transducer HtrXIII 6542 probable transposase (insertion sequence IS861) 6544 ABC transporter, ATP-binding/permease protein 6550 ABC transporter, ATP-binding/permease protein 6560 Serine hydroxymethyltransferase 6568 HemK protein (hemK) 6572 peptide chain release factor 1 (prfA) 6576 thymidine kinases 6580 4-oxalocrotonate tautomerase (dmpI) 6588 oxidoreductase 6594 oxidoreductase 6600 formate/nitrite transporter family protein 6608 xanthine permease (pbuX) 6612 xanthine phosphoribosyltransferase (xpt) 6616 guanosine monophosphate reductase (guaC) 6620 drug resistance transporter, EmrB/QacA subfamily 6622 oxidoreductase 6624 Kup system potassium uptake protein (kup) 6636 O-methyltransferase 6642 oligoendopeptidase F (pepF) 6646 competence protein CoiA (coiA) 6650 major facilitator superfamily protein superfamily 6652 ribosomal small subunit pseudouridine synthase A (rsuA) 6658 glucosamine-6-phosphate isomerase (nagB) 6662 nodulin-related protein, truncation 6664 S-adenosylmethioninetRNA ribosyltransferase-isomerase (queA) 6674 permease, GntP family 6684 6-phospho-beta-glucosidase (bglA) 6686 PTS system, beta-glucosides-specific IIABC components 6688 transcription antiterminator Lict (licT) 6704 esterase 6706 sugar-binding transcriptional repressor, Lacl family 6708 hydrolase, haloacid dehalogenase-like family 6712 DNA internalization-related competence protein ComEC/Rec2 6716 competence protein CelA (celA) 6720 acyltransferase family protein 6732 ATP-dependent RNA helicase DeaD (deaD) 6736 lipoprotein, YaeC family 6738 ABC transporter, permease protein 6752 diacylglycerol kinase (dgkA) 6768 formamidopyrimidine-DNA glycosylase (mutM) 6776 epidermin immunity protein F 6788 glycyl-tRNA synthetase, beta subunit (glyS) 6790 acyl carrier protein phosphodiesterase 6800 SsrA-binding protein (smpB) 6822 D-alanine--D-alanine ligase 6824 recombination protein RecR (recR) 6830 penicillin-binding protein 2b 6832 phosphoglycerate mutase (gpmA) 6836 triosephosphate isomerase (tpiA) 6856 phosphoglycerate mutase family protein 6860 D-alanyl-D-alanine carboxypeptidase family 6864 autolysin 6868 heat-inducible transcription repressor HrcA (hrcA) 6872 heat shock protein GrpE (grpE) 6876 chaperone protein dnak 6880 dnaJ protein (dnaJ) 6884 transcriptional regulator, gntR family domain protein 6888 tRNA pseudouridine synthase A (truA) 6892 phosphomethylpyrimidine kinase (thiD) 6910 galactose-6-phosphate isomerase, LacA subunit (lacA) 6922 tagatose 1,6-diphosphate aldolase (lacD) 6932 sugar ABC transporter, ATP-binding protein (msmK) 6936 glucan 1,6-alpha-glucosidase (dexB) 6940 UDP-glucose 4-epimerase (galE) 6942 response regulator (citB) 6950 citrate carrier protein (citS) 6954 malate oxidoreductase (tme) 6958 bacterocin transport accessory protein 6976 transposase family protein (orfA) 6980 pXO1-128 6986 adhesion lipoprotein (lmb) 6994 DNA-directed RNA polymerase, alpha subunit (rpoA) 6998 ribosomal protein L17 (rplQ) 7040 probable dna-directed rna polymerase delta subunit 7044 CTP synthase (pyrG) 7058 bacteriocin transport accessory protein 7074 translation initiation factor IF-3 (infC) 7100 adenosine deaminase 8468 preprotein translocase, SecE subunit 8476 antigen, 67 kDa 8486 Lipase/Acylhydrolase 8492 peptide ABC transporter, permease protein (oppB) 8494 competence protein CglB (cglB) 8502 peptide ABC transporter, peptide-binding protein 8504 oxidoreductase 8510 amino acid ABC transporter, permease protein (opuBB) 8522 abc transporter atp-binding protein ybhf 8530 glycerol-3-phosphate dehydrogenase (NAD(P)+) (gpsA) 8538 sugar ABC transporter, sugar-binding protein 8544 secreted 45 kd protein (usp45) 8556 phosphoglycerate mutase family protein 8566 glycosyl hydrolase, family 3 8576 N-acetylmuramoyl-L-alanine amidase 8596 sensory box histidine kinase (withHAMPandPASd) 8608 aminoglycoside 6-adenylyltransferase 8622 iron compound ABC transporter, permease protein (sirB) 8636 phosphate ABC transporter, permease protein (pstC-2) 8650 branched-chain amino acid transport system II carrier protein (brnQ) 8658 PTS system, IID component 8662 replisome organiser-related protein 8674 alkaline amylopullulanase 8676 exfoliative toxin A 8690 glycerol uptake facilitator protein (glpF) 8698 ABC transporter, ATP-binding protein 8706 CDP-diacylglycerol--glycerol-3-phosphate 3-phosphatidyltransferase (pgs 8708 cobalt transport protein 8730 integral membrane protein 8734 yadS protein 8736 cell wall surface anchor family protein 8748 polysaccharide biosynthesis protein 8752 glycosyl transferase domain protein 8764 endopeptidase O 8770 beta-ketoacyl-acyl carrier protein synthase II 8772 ABC transporter, ATP-binding protein 8776 penicillin-binding protein 8778 cell wall surface anchor family protein 8780 cell wall surface anchor family protein 8786 LPXTG-motif cell wall anchor domain protein 8788 6-aminohexanoate-cyclic-dimer hydrolase 8796 NLP/P60 family protein 8802 DNA/RNA non-specific endonuclease 8806 hydroxyethylthiazole kinase (thiM) 8826 PTS system component 8832 sugar ABC transporter, permease protein 8836 potassium uptake protein, Trk family (trkA) 8850 lemA protein (lemA) 8856 cobalt transport protein 8882 spermidine/putrescine ABC transporter, spermidine/putrescine-binding pr 8884 spermidine/putrescine ABC transporter, permease protein (potC) 8906 ABC transporter, substrate-binding protein 8908 lipoprotein 8916 sensor histidine kinase 8930 TrsK-like protein (traK) 8936 R5 protein 8962 chromosome assembly protein homolog 8978 ribose ABC transporter, permease protein (rbsC) 8980 permease 8982 sensor histidine kinase (arlS) 8986 hydrolase, haloacid dehalogenase-like family (gph) 8994 dephospho-CoA kinase 8996 oxalateformate antiporter 9004 sensory box protein 9006 host cell surface-exposed lipoprotein 9012 PAP2 family protein 9034 GtrA family protein 9050 lipoprotein signal peptidase (lspA) 9280 alcohol dehydrogenase, zinc-containing (adh) 9284 trigger factor (tig) 9290 fructose-bisphosphate aldolase (fba) 9292 DAK2 domain protein 9296 oligopeptide ABC transporter, permease protein 9298 N-acetylglucosamine-6-phosphate deacetylase (nagA) 9300 transcriptional regulator, DeoR family (lacR) 9302 PTS system, mannose-specific IIC component (manM) 9306 Phosphoglucose isomerase 9310 aspartate--ammonia ligase (asnA) 9312 amino acid ABC transporter, ATP-binding protein 9314 DNA-binding protein HU (hup) 9316 DHH subfamily 1 protein 9318 chloride channel 9320 integrase (int) 9324 DNA/RNA non-specific endonuclease 9326 PTS system component 9328 cell division protein, FtsW/RodA/SpoVE family (ftsW) 9330 LPXTG-motif cell wall anchor domain protein 9332 peptide chain release factor 3 (prfC) 9334 ABC transporter, ATP-binding protein 9336 superoxide dismutase [mn-fe] 9340 phenylalanyl-tRNA synthetase, alpha subunit (pheS) 9342 amino acid ABC transporter, permease protein 9344 phosphate ABC transporter, phosphate-binding protein (pstS) 9346 NOL1/NOP2/sun family protein (sun) 9348 Abortive infection protein family 9350 permease 9352 N-acetylmuramoyl-L-alanine amidase domain protein (usp45) 9354 ABC transporter, ATP-binding protein 9356 phosphoglucomutase (pgm) 9358 oxidoreductase, short chain dehydrogenase/reductase family 9360 phosphate acetyltransferase 9362 gls24 protein 9364 ribosomal protein S1 (rpsA) 9368 dTDP-glucose 4,6-dehydratase (rfbB) 9370 excinuclease ABC, C subunit (uvrC) 9372 MATE efflux family protein 9378 amino acid permease (rocE) 9380 DNA-binding response regulator TrcR (trcR) 9382 16S rRNA processing protein RimM (rimM) 9384 transcriptional regulator 9388 ribosomal protein L20 (rplT) 9394 sugar-binding transcriptional repressor, Lacl family (malR) 9396 proton/peptide symporter family protein 9398 amino acid permease 9400 exoribonuclease, VacB/Rnb family (vacB) 9402 multi-drug resistance efflux pump (pmrA) 9404 adhesion lipoprotein (psaA) 9406 iron-dependent transcriptional regulator (sirR) 9410 branched-chain amino acid ABC transporter, amino acid-binding protein ( 9412 amino acid permease 9414 SpoU rRNA Methylase family protein 9416 sodium/dicarboxylate symporter (gltP-2) 9418 branched-chain amino acid transport system II carrier protein (brnQ) 9420 alcohol dehydrogenase, zinc-containing 9422 aminotransferase, class I (aspB) 9424 ribosomal protein S6 (rpsF) 9426 A/G-specific adenine glycosylase (mutY) 9428 acid phosphatase (olpA) 9430 ribosomal protein S12 (rpsL) 9434 microcin immunity protein MccF (mccF-1) 9436 undecaprenyl diphosphate synthase (uppS) 9438 preprotein translocase, YajC subunit (yajC) 9440 chaperonin, 10 kDa (groES) 9444 YitT family protein 9446 serine protease (htrA) 9448 ribose-phosphate pyrophosphokinase (prsA) 9450 aromatic amino acid aminotransferase (araT) 9452 Recombination protein O (recO) 9454 Abortive infection protein family 9456 fatty acid/phospholipid synthesis protein PlsX (plsX) 9458 acyl carrier protein (acpP) 9462 phosphoribosylaminoimidazole carboxylase, ATPase subunit (purK) 9464 alcohol dehydrogenase, iron-containing 9466 ribosomal protein L18 (rplR) 9468 preprotein translocase, SecY subunit 9470 transcriptional regulator ComX1 (comX1) 9472 deoxyuridine 5′-triphosphate nucleotidohydrolase (dut) 9478 sugar-binding transcriptional regulator, Lacl family (rbsR) 9480 SPFH domain/Band 7 family 9488 zinc ABC transporter, permease protein (adcB) 9492 abortive infection protein 9494 hydrolase, haloacid dehalogenase-like family 9496 response regulator (lytT) 9500 transketolase, C-terminal subunit 9502 polyribonucleotide nucleotidyltransferase (pnp) 9504 serine O-acetyltransferase (cysE) 9508 ribosomal protein L13 (rplM) 9510 replication initiation protein 9518 amino acid ABC transporter, amino acid-binding protein 9522 glycyl-tRNA synthetase, alpha subunit (glyQ) 9524 NADH oxidase 9528 transketolase (tkt) 9534 penicillin-binding protein 1A (pbp1A) 9536 cell division protein DivIVA (divIVA) 9538 sensor histidine kinase 9540 serine/threonine protein kinase (pknB) 9542 transcriptional regulator 9544 PTS system, IIA component (lacF) 9546 glycerol dehydrogenase (gldA) 9548 aspartate kinase (thrA) 9550 enoyl-CoA hydratase/isomerase family protein 9552 acyl carrier protein (acpP) 9564 ABC transporter, ATP-binding protein 9566 N utilization substance protein A (nusA) 9568 ribosome-binding factor A (rbfA) 9570 Cof family protein 9572 CoA binding domain protein (b0965) 9574 transcriptional regulator, Fur family 9578 queuine tRNA-ribosyltransferase (tgt) 9580 ribonuclease P protein component (rnpA) 9582 serine protease, subtilase family 9584 glycosyl transferase domain protein 9586 transcriptional activator, AraC family 9588 transcriptional regulator, TetR family 9590 transcriptional regulator, AraC family 9594 surface protein Rib 9596 transposase, mutator family 9600 acetyltransferase, GNAT family 9602 Transposase, Mutator family 9606 UDP-sugar hydrolase 9610 anthranilate synthase component II (trpG) 9612 biotin synthetase (bioB) 9616 UDP-N-acetylmuramoylalanine--D-glutamate ligase (murD) 9618 ylmF protein (ylmF) 9620 amino acid ABC transporter, permease protein 9622 phosphoglucomutase (pgm) 9624 YjeF-related protein, C-terminus 9626 FemAB family protein (fibA) 9628 Cof family protein 9630 cell division ABC transporter, permease protein FtsX (ftsX) 9632 oxidoreductase, short-chain dehydrogenase/reductase family (fabG) 9634 aspartate aminotransferase (aspC) 9638 ribosomal protein L31 (rpmE) 9640 nrdI protein (nrdI) 9642 ribosomal protein L19 (rplS) 9644 bacteriophage L54a, repressor protein 9646 bacteriophage L54a, antirepressor 9652 single-strand binding protein (ssb) 9660 pneumococcal surface protein A 9666 DNA-binding response regulator (vncR) 9668 transposase OrfAB, subunit B 9670 cell division protein, FtsW/RodA/SpoVE family (rodA) 9672 DNA gyrase, B subunit (gyrB) 9674 3-phosphoshikimate 1-carboxyvinyltransferase (aroA) 9676 RNA methyltransferase, TrmA family 9680 transcriptional regulator, AraC family 9682 ABC transporter, ATP-binding protein 9690 CylJ 9696 permease 9698 regulatory protein 9700 carbohydrate kinase, pfkB family 9702 beta-glucuronidase 9704 2-deydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldo 9706 3-oxoacyl-(acyl-carrier-protein) reductase 9708 catabolite control protein A (ccpA) 9712 ribonuclease III (rnc) 9714 SMC family, C-terminal domain family 9718 S1 RNA binding domain protein 9722 prolipoprotein diacylglyceryl transferase (lgt) 9724 riboflavin synthase, alpha subunit (ribE) 9726 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II (ri 9728 lysyl-tRNA synthetase (lysS) 9734 Transposase subfamily 9738 translation elongation factor Tu (tuf) 9740 UDP-N-acetylmuramoylalanyl-D-glutamyl-2,6-diaminopimelate--D-alanyl-D-a 9746 Glutathione S-transferases domain protein 9754 Ribonucleotide reductases 9756 biotin--acetyl-CoA-carboxylase ligase 9760 Uncharacterized protein family SNZ family 9762 methionine aminopeptidase, type I (map) 9764 DNA ligase, NAD-dependent (ligA) 9766 glucose-1-phosphate adenylyltransferase (glgC) 9768 UDP-N-acetylglucosamine 1-carboxyvinyltransferase (murA) 9770 acetyltransferase, GNAT family 9772 exonuclease RexA (rexA) 9774 tRNA modification GTPase TrmE (trmE) 9776 ABC transporter, ATP-binding protein 9778 pyruvate dehydrogenase complex, E1 component, pyruvate dehydrogenase al 9782 Mur ligase family protein 9786 HD domain protein 9788 translation elongation factor G (fusA) 9796 pyruvate kinase (pyk) 9798 Signal peptidase I 9802 cytidine deaminase (cdd) 9804 sugar ABC transporter, ATP-binding protein 9806 sugar ABC transporter, permease protein 9808 acetyltransferase, GNAT family 9810 ABC transporter, permease protein 9812 SatD 9814 Helix-turn-helix domain, fis-type protein 9816 phosphate ABC transporter, ATP-binding protein (pstB-1) 9818 tRNA pseudouridine synthase B (truB) 9820 Acetyltransferase (GNAT) family 9822 DNA topoisomerase I (topA) 9824 ribonuclease HII (rnhB) 9830 orotidine 5′-phosphate decarboxylase (pyrF) 9832 aspartate-semialdehyde dehydrogenase (asd) 9836 pantothenate metabolism flavoprotein (dfp) 9840 Sua5/YciO/YrdC/YwlC family protein 9844 thiamine biosynthesis protein ApbE 9846 Domain of unknown function 9848 DNA repair protein RadC (radC) 9850 glycosyl hydrolase, family 1 (bglA) 9852 Cof family protein (b0844) 9854 spermidine/putrescine ABC transporter, permease protein (potH) 9856 folylpolyglutamate synthase (folC) 9858 homoserine dehydrogenase (hom) 9860 succinate-semialdehyde dehydrogenase (gabD-1) 9862 membrane protein 9864 ATP-dependent DNA helicase PcrA (pcrA) 9866 uracil permease (uraA) 9868 sodiumalanine symporter family protein 9878 capsular polysaccharide biosynthesis protein Cps4B (cps4B) 9880 transcriptional regulator, LysR family 9882 CpslaS 9884 chloride channel protein 9886 tributyrin esterase (estA) 9888 ABC transporter, ATP-binding protein (potA) 9890 alpha-acetolactate decarboxylase (budA) 9892 TPR domain protein 9896 metallo-beta-lactamase superfamily protein 9898 tRNA delta(2)-isopentenylpyrophosphate transferase (miaA) 9902 glycerophosphoryl diester phosphodiesterase 9904 transposase OrfAB, subunit B 9906 IS3-Spn1, transposase 9908 transposase OrfAB, subunit B (orfB) 9910 reverse transcriptase 9916 transposase OrfAB, subunit B 9918 integrase, phage family (int) 9920 transcription regulator 9922 TnpA 9926 structural gene for ultraviolet resistance (uvra) 9930 Helicases conserved C-terminal domain protein 9932 abortive infection bacteriophage resistance protein (abiEi) 9944 ribosomal protein L7/L12 (rplL) 9948 ATP-dependent Clp protease, ATP-binding subunit ClpX (clpX) 9950 dihydrofolate reductase (folA) 9952 hemolysin 9954 transcriptional regulator, MarR family 9958 polyA polymerase family protein 9960 PTS system, fructose specific IIABC components (fruA-1) 9962 lactose phosphotransferase system repressor (lacR) 9964 choline binding protein D (cbpD) 9968 pyrimidine operon regulatory protein (pyrR) 9970 ribosomal large subunit pseudouridine synthase D (rluD) 9972 thiamine biosynthesis protein ThiI (thiI) 9974 3-dehydroquinate dehydratase, type I (aroD) 9976 iron compound ABC transporter, ATP-binding protein (fepC) 9980 transcriptional regulator 9982 glycosyl transferase domain protein 9984 Cps9H 9988 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (ispD) 9990 licD1 protein (licD1) 9996 large conductance mechanosensitive channel protein (mscL) 10000 maltose ABC transporter, maltose-binding protein 10004 nucleotide sugar synthetase-like protein 10006 transcriptional regulator 10008 oxidoreductase, aldo/keto reductase family 10010 NAD(P)H-flavin oxidoreductase 10016 transcriptional regulator MutR 10018 GTP-binding protein Era (era) 10022 peptide methionine sulfoxide reductase (msrA) 10026 peptide ABC transporter, ATP-binding protein 10028 peptide ABC transporter, ATP-binding protein (amiE) 10030 peptide ABC transporter, peptide-binding protein 10032 transposase, IS30 family 10034 transcriptional regulator, LysR family 10036 spoE family protein (ftsK) 10044 methionyl-tRNA synthetase (metG) 10046 D-isomer specific 2-hydroxyacid dehydrogenase family protein (serA) 10048 acetyltransferase, GNAT family 10050 phosphoserine aminotransferase (serC) 10054 thymidylate kinase (tmk) 10060 branched-chain amino acid ABC transporter, permease protein (livH) 10062 ATP-dependent Clp protease, proteolytic subunit ClpP (clpP) 10064 uracil phosphoribosyltransferase (upp) 10066 potassium uptake protein, Trk family (trkH) 10068 glutamate racemase (murI) 10070 membrane protein 10072 HD domain protein 10074 Acylphosphatase 10076 spoIIIJ family protein 10078 acetyltransferase, GNAT family 10080 glucose-inhibited division protein B (gidB) 10082 potassium uptake protein, Trk family 10084 ABC transporter, permease protein 10088 isochorismatase family protein 10092 haloacid dehalogenase-like hydrolase superfamily 10094 membrane protein 10096 glutamyl-tRNA(Gln) amidotransferase, B subunit (gatB) 10098 CBS domain protein protein 10100 transcriptional regulator (codY) 10102 universal stress protein family 10104 L-asparaginase (ansA) 10106 oxidoreductase, aldo/keto reductase 2 family 10108 preprotein translocase, SecA subunit (secA) 10112 excinuclease ABC, A subunit (uvrA) 10114 magnesium transporter, CorA family (corA) 10116 thioredoxin (trx) 10118 MutS2 family protein (mutS2) 10122 DNA-damage inducible protein P (dinP) 10124 formate acetyltransferase (pfl) 10126 transcriptional regulator, Crp family 10128 transport ATP-binding protein CydC 10138 ribosomal-protein-alanine acetyltransferase (rimI) 10140 hydrolase 10144 D-alanine-activating enzyme (dltA) 10148 carbohydrate kinase, FGGY family 10150 transaldolase 10160 Helix-turn-helix domain protein 10164 single-strand binding protein (ssb) 10166 type II DNA modification methyltransferase Spn5252IP (spn5252IMP) 10174 integrase, phage family 10178 Cyclic nucleotide-binding domain protein 10180 transcriptional regulator, MarR family 10182 prolyl-tRNA synthetase (proS) 10184 leucine-rich protein 10186 lacX protein, truncation (lacX) 10188 tagatose-6-phosphate kinase (lacC) 10190 galactose-6-phosphate isomerase, LacB subunit (lacB) 10192 neuraminidase 10198 Histidine kinase-, DNA gyrase B-, phytochrome-like ATPase domain protei 10200 ABC transporter, ATP-binding protein 10202 PTS system, IIABC components (ptsG) 10204 phosphate regulon response regulator PhoB (phoB) 10212 Uncharacterized ACR, COG2161 subfamily 10216 abortive phage resistance protein 10222 TnpA 10226 acetyltransferase, GNAT family 10230 ABC transporter domain protein 10234 5-methyltetrahydropteroyltriglutamate--homocysteine methyltransferase ( 10236 branched-chain amino acid transport protein AzlC (azlC) 10240 DNA-binding response regulator (srrA) 10242 leucyl-tRNA synthetase (leuS) 10246 NupC family protein 10248 transcriptional regulator, GntR family 10252 glyoxalase family protein 10254 anaerobic ribonucleoside-triphosphate reductase (nrdD) 10256 competence-induced protein Ccs4 10262 competence/damage-inducible protein CinA (cinA) 10264 DNA-3-methyladenine glycosylase I (tag) 10268 DNA mismatch repair protein HexB (hexB) 10270 arginine repressor (argR) 10272 arginyl-tRNA synthetase (argS) 10274 aspartyl-tRNA synthetase (aspS) 10276 histidyl-tRNA synthetase (hisS) 10280 AGR_pAT_51p 10286 hydrolase, alpha/beta hydrolase fold family 10288 phage infection protein 10290 Glucose inhibited division protein A (gidA) 10292 tRNA (5-methylaminomethyl-2-thiouridylate)-methyltransferase (trmU) 10296 arginine/ornithine antiporter (arcD) 10298 chromosomal replication initiator protein DnaA (dnaA) 10302 peptidyl-tRNA hydrolase (pth) 10310 phosphotyrosine protein phosphatase 10316 ribosomal protein L36 (rpmJ) 10318 ribosomal protein S13/S18 (rpsM) 10328 L-lactate dehydrogenase (ldh) 10330 ribosomal protein L28 (rpmB) 10362 RNA polymerase sigma-70 factor, ECF subfamily 10384 BioY family protein 10386 AtsA/ElaC family protein 10388 cytidine/deoxycytidylate deaminase family protein 10394 phosphorylase, Pnp/Udp family 10396 transcriptional regulator, MerR family 10402 methyltransferase (ubiE) 10412 type IV prepilin peptidase 10416 ylmG protein (ylmG) 10444 transposase OrfAB, subunit B 10446 IS150-like transposase 10452 Bacterial regulatory proteins, tetR family domain protein 10454 cell wall surface anchor family protein, authentic frameshift (clfB) 10456 transposase OrfAB, subunit A (orfA) 10460 chaperonin, 33 kDa (hslO) 10472 (3R)-hydroxymyristoyl-(acyl-carrier-protein) dehydratase (fabZ) 10482 sprT protein 10490 transcriptional regulator, MarR family 10498 transcriptional regulator 10504 glycogen biosynthesis protein GlgD (glgD) 10536 ribonucleoside-diphosphate reductase, alpha subunit, truncation (nrdD) 10538 LPXTG-motif cell wall anchor domain 10550 membrane protein 10554 arsenate reductase (arsC) 10564 transposase, authentic frameshift 10570 transposase OrfAB, subunit A (orfA) 10574 Tn5252, Orf 9 protein 10580 IS3-Spn1, transposase 10584 transcriptional regulator, ArsR family 10628 ribosomal protein L35 (rpmI) 10630 cytidylate kinase (cmk) 10636 MutT/nudix family protein 10644 preprotein translocase, SecG subunit 10680 ribosomal protein S18 (rpsR) 10682 single-strand binding protein (ssb) 10692 glyceraldehyde 3-phosphate dehydrogenase (gap) 10694 translation elongation factor G (fusA) 10696 ribosomal protein S7 (rpsG) 10704 phosphinothricin N-acetyltransferase (pat) 10730 nrdI protein (nrdI) 10732 accessory gene regulator protein C (blpH) 10744 rhodanese family protein (pspE) 10746 cAMP factor 10758 competence/damage-inducible protein CinA (cinA) 10770 transcriptional regulator, ArgR family (argR) 10772 FliP family family 10794 peptide ABC transporter, peptide-binding protein 10800 ribosomal protein S21 (rpsU) 10802 transposase, IS30 family 10816 mucin 2 precursor, intestinal 10854 SV40-transformed marker protein pG1-related protein 10856 SV40-transformed marker protein pG1-related protein 10858 SV40-transformed marker protein pG1-related protein 10860 SV40-transformed marker protein pG1-related protein 10862 SV40-transformed marker protein pG1-related protein 10864 SV40-transformed marker protein pG1-related protein 10866 SV40-transformed marker protein pG1-related protein 10910 transcriptional regulator 10920 ribosomal protein S11 (rpsK) 10922 elaA protein 10926 5-formyltetrahydrofolate cyclo-ligase family protein 10938 inositol monophosphatase family protein 10940 amino acid ABC transporter, amino acid-binding protein (artI) 10944 Holliday junction DNA helicase RuvB (ruvB) 10946 D-alanyl-D-alanine carboxypeptidase (dacA) 10948 lipoprotein (bmpD) 10950 peptidase, U32 family family 10952 protease maturation protein 10954 glutamyl-tRNA synthetase (gltX) 10956 GTP-binding protein LepA (lepA) 10960 translation initiation factor if-2 10962 phosphoenolpyruvate carboxylase (ppc) 10964 calcium E1-E2-type ATPase 10966 serine protease, subtilase family

Exemplary Sequences  SEQ ID NO: 4209  atgaataagc catattcaat aggccttgac atcggtacta attccgtcgg atggagcatt   60  attacagatg attataaagt acctgctaag aagatgagag ttttagggaa cactgataaa  120  gaatatatta agaagaatct cataggtgct ctgctttttg atggcgggaa tactgctgca  180  gatagacgct tgaagcgaac tgctcgtcgt cgttatacac gtcgtagaaa tcgtattcta  240  tatttacaag aaatttttgc agaggaaatg agtaaagttg atgatagttt ctttcatcga  300  ttagaggatt cttttctagt tgaggaagat aagagaggga gcaagtatcc tatctttgca  360  acattgcagg aagagaaaga ttatcatgaa aaattttcga caatctatca tttgagaaaa  420  gaattagctg acaagaaaga aaaagcagac cttcgtctta tttatattgc tctagctcat  480  atcattaaat ttagagggca tttcctaatt gaggatgata gctttgatgt caggaataca  540  gacatttcaa aacaatatca agatttttta gaaatcttta atacaacttt tgaaaataat  600  gatttgttat ctcaaaacgt tgacgtagag gcaatactaa cagataagat tagcaagtct  660  gcgaagaaag atcgtatttt agcgcagtat cctaaccaaa aatctactgg catttttgca  720  gaatttttga aattgattgt cggaaatcaa gctgacttca agaaatattt caatttggag  780  gataaaacgc cgcttcaatt cgctaaggat agctacgatg aagatttaga aaatcttctt  840  ggacagattg gtgatgaatt tgcagactta ttctcagcag cgaaaaagtt atatgatagt  900  gtccttttgt ctggcattct tacagtaatc gacctcagta ccaaggcgcc actttcagct  960  tctatgattc agcgttatga tgaacataga gaggacttga aacagttaaa acaattcgta 1020  aaagcttcat tgccggaaaa atatcaagaa atatttgctg attcatcaaa agatggctac 1080  gctggttata ttgaaggtaa aactaatcaa gaagcttttt ataaatacct gtcaaaattg 1140  ttgaccaagc aagaagatag cgagaatttt cttgaaaaaa tcaagaatga agatttcttg 1200  agaaaacaaa ggacctttga taatggctca attccacacc aagtccattt gacagagctg 1260  aaagctatta tccgccgtca atcagaatac tatcccttct tgaaagagaa tcaagatagg 1320  attgaaaaaa tccttacctt tagaattcct tattatatcg ggccactagc acgtgagaag 1380  agtgattttg catggatgac tcgcaaaaca gatgacagta ttcgaccttg gaattttgaa 1440  gacttggttg ataaagaaaa atctgcggaa gcttttatcc atcgtatgac caacaatgat 1500  ttttatcttc ctgaagaaaa agttttacca aagcatagtc ttatttatga aaaatttacg 1560  gtctataatg agttgactaa ggttagatat aaaaatgagc aaggtgagac ttattttttt 1620  gatagcaata ttaaacaaga aatctttgat ggagtattca aggaacatcg taaggtatcc 1680  aagaagaagt tgctagattt tctggctaaa gaatatgagg agtttaggat agtagatgtt 1740  attggtctag ataaagaaaa taaagctttc aacgcctcat tgggaactta ccacgatctc 1800  gaaaaaatac tagacaaaga ttttctagat aatccagata atgagtctat tctggaagat 1860  atcgtccaaa ctctaacatt atttgaagac agagaaatga ttaagaagcg tcttgaaaac 1920  tataaagatc tttttacaga gtcacaacta aaaaaactct atcgtcgtca ctatactggc 1980  tggggacgat tgtctgctaa gttaatcaat ggtattcgag ataaagagag tcaaaaaaca 2040  atcttggact atcttattga tgatggtaga tctaatcgca actttatgca gttgataaat 2100  gatgatggtc tatctttcaa atcaattatc agtaaggcac aggctggtag tcattcagat 2160  aatctaaaag aagttgtagg tgagcttgca ggtagccctg ctattaaaaa gggaattcta 2220  caaagtttga aaattgttga tgagcttgtt aaagtcatgg gatacgaacc tgaacaaatt 2280  gtggttgaga tggcgcgtga gaatcaaaca acaaatcaag gtcgtcgtaa ctctcgacaa 2340  cgctataaac ttcttgatga tggcgttaag aatctagcta gtgacttgaa tggcaatatt 2400  ttgaaagaat atcctacgga taatcaagcg ttgcaaaatg aaagactttt cctttactac 2460  ttacaaaacg gaagagatat gtatacaggg gaagctctag atattgacaa tttaagtcaa 2520  tatgatattg accacattat tcctcaagct ttcataaaag atgattctat tgataatcgt 2580  gttttggtat catctgctaa aaatcgtgga aagtcagatg atgttcctag ccttgaaatt 2640  gtaaaagatt gtaaagtttt ctggaaaaaa ttacttgatg ctaagttaat gagtcagcgt 2700  aagtatgata atttgactaa ggcagagcgc ggaggcctaa cttccgatga taaggcaaga 2760  tttatccaac gtcagttggt tgagacacga caaattacca agcatgttgc ccgtatcttg 2820  gatgaacgct ttaataatga gcttgatagt aaaggtagaa ggatccgcaa agttaaaatt 2880  gtaaccttga agtcaaattt ggtttcaaat ttccgaaaag aatttggatt ctataaaatt 2940  cgtgaagtta acaattatca ccatgcacat gatgcctatc ttaatgcagt agttgctaaa 3000  gctattctaa ccaaatatcc tcagttagag ccagaatttg tctacggcga ctatccaaaa 3060  tataatagtt acaaaacgcg taaatccgct acagaaaagc tatttttcta ttcaaatatt 3120  atgaacttct ttaaaactaa ggtaacttta gcggatggaa ccgttgttgt aaaagatgat 3180  attgaagtta ataatgatac gggtgaaatt gtttgggata aaaagaaaca ctttgcgaca 3240  gttagaaaag tcttgtcata ccctcagaac aatatcgtga agaagacaga gattcagaca 3300  ggtggtttct ctaaggaatc aatcttggcg catggtaact cagataagtt gattccaaga 3360  aaaacgaagg atatttattt agatcctaag aaatatggag gttttgatag tccgatagta 3420  gcttactctg ttttagttgt agctgatatc aaaaagggta aagcacaaaa actaaaaaca 3480  gttacggaac ttttaggaat taccatcatg gagaggtcca gatttgagaa aaatccatca 3540  gctttccttg aatcaaaagg ctatttaaat attagggctg ataaactaat tattttgccc 3600  aagtatagtc tgttcgaatt agaaaatggg cgtcgtcgat tacttgctag tgctggtgaa 3660  ttacaaaaag gtaatgagct agccttacca acacaattta tgaagttctt ataccttgca 3720  agtcgttata atgagtcaaa aggtaaacca gaggagattg agaagaaaca agaatttgta 3780  aatcaacatg tctcttattt tgatgacatc cttcaattaa ttaatgattt ttcaaaacga 3840  gttattctag cagatgctaa tttagagaaa atcaataagc tttaccaaga taataaggaa 3900  aatatatcag tagatgaact tgctaataat attatcaatc tatttacttt taccagtcta 3960  ggagctccag cagcttttaa attttttgat aaaatagttg atagaaaacg ctatacatca 4020  actaaagaag tacttaattc taccctaatt catcaatcta ttactggact ttatgaaaca 4080  cgtattgatt tgggtaagtt aggagaagat 4110  SEQ ID NO: 4210  Met Asn Lys Pro Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val  1               5                   10                  15  Gly Trp Ser Ile Ile Thr Asp Asp Tyr Lys Val Pro Ala Lys Lys Met              20                  25                  30  Arg Val Leu Gly Asn Thr Asp Lys Glu Tyr Ile Lys Lys Asn Leu Ile          35                  40                  45  Gly Ala Leu Leu Phe Asp Gly Gly Asn Thr Ala Ala Asp Arg Arg Leu      50                  55                  60  Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Arg Asn Arg Ile Leu  65                  70                  75                  80  Tyr Leu Gln Glu Ile Phe Ala Glu Glu Met Ser Lys Val Asp Asp Ser                  85                  90                  95  Phe Phe His Arg Leu Glu Asp Ser Phe Leu Val Glu Glu Asp Lys Arg              100                 105                 110  Gly Ser Lys Tyr Pro Ile Phe Ala Thr Leu Gln Glu Glu Lys Asp Tyr          115                 120                 125  His Glu Lys Phe Ser Thr Ile Tyr His Leu Arg Lys Glu Leu Ala Asp      130                 135                 140  Lys Lys Glu Lys Ala Asp Leu Arg Leu Ile Tyr Ile Ala Leu Ala His  145                 150                 155                 160  Ile Ile Lys Phe Arg Gly His Phe Leu Ile Glu Asp Asp Ser Phe Asp                  165                 170                 175  Val Arg Asn Thr Asp Ile Ser Lys Gln Tyr Gln Asp Phe Leu Glu Ile              180                 185                 190  Phe Asn Thr Thr Phe Glu Asn Asn Asp Leu Leu Ser Gln Asn Val Asp          195                 200                 205  Val Glu Ala Ile Leu Thr Asp Lys Ile Ser Lys Ser Ala Lys Lys Asp      210                 215                 220  Arg Ile Leu Ala Gln Tyr Pro Asn Gln Lys Ser Thr Gly Ile Phe Ala  225                 230                 235                 240  Glu Phe Leu Lys Leu Ile Val Gly Asn Gln Ala Asp Phe Lys Lys Tyr                  245                 250                 255  Phe Asn Leu Glu Asp Lys Thr Pro Leu Gln Phe Ala Lys Asp Ser Tyr              260                 265                 270  Asp Glu Asp Leu Glu Asn Leu Leu Gly Gln Ile Gly Asp Glu Phe Ala          275                 280                 285  Asp Leu Phe Ser Ala Ala Lys Lys Leu Tyr Asp Ser Val Leu Leu Ser      290                 295                 300  Gly Ile Leu Thr Val Ile Asp Leu Ser Thr Lys Ala Pro Leu Ser Ala  305                 310                 315                 320  Ser Met Ile Gln Arg Tyr Asp Glu His Arg Glu Asp Leu Lys Gln Leu                  325                 330                 335  Lys Gln Phe Val Lys Ala Ser Leu Pro Glu Lys Tyr Gln Glu Ile Phe              340                 345                 350  Ala Asp Ser Ser Lys Asp Gly Tyr Ala Gly Tyr Ile Glu Gly Lys Thr          355                 360                 365  Asn Gln Glu Ala Phe Tyr Lys Tyr Leu Ser Lys Leu Leu Thr Lys Gln      370                 375                 380  Glu Asp Ser Glu Asn Phe Leu Glu Lys Ile Lys Asn Glu Asp Phe Leu  385                 390                 395                 400  Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Val His                  405                 410                 415  Leu Thr Glu Leu Lys Ala Ile Ile Arg Arg Gln Ser Glu Tyr Tyr Pro              420                 425                 430  Phe Leu Lys Glu Asn Gln Asp Arg Ile Glu Lys Ile Leu Thr Phe Arg          435                 440                 445  Ile Pro Tyr Tyr Ile Gly Pro Leu Ala Arg Glu Lys Ser Asp Phe Ala      450                 455                 460  Trp Met Thr Arg Lys Thr Asp Asp Ser Ile Arg Pro Trp Asn Phe Glu  465                 470                 475                 480  Asp Leu Val Asp Lys Glu Lys Ser Ala Glu Ala Phe Ile His Arg Met                  485                 490                 495  Thr Asn Asn Asp Phe Tyr Leu Pro Glu Glu Lys Val Leu Pro Lys His              500                 505                 510  Ser Leu Ile Tyr Glu Lys Phe Thr Val Tyr Asn Glu Leu Thr Lys Val          515                 520                 525  Arg Tyr Lys Asn Glu Gln Gly Glu Thr Tyr Phe Phe Asp Ser Asn Ile      530                 535                 540  Lys Gln Glu Ile Phe Asp Gly Val Phe Lys Glu His Arg Lys Val Ser  545                 550                 555                 560  Lys Lys Lys Leu Leu Asp Phe Leu Ala Lys Glu Tyr Glu Glu Phe Arg                  565                 570                 575  Ile Val Asp Val Ile Gly Leu Asp Lys Glu Asn Lys Ala Phe Asn Ala              580                 585                 590  Ser Leu Gly Thr Tyr His Asp Leu Glu Lys Ile Leu Asp Lys Asp Phe          595                 600                 605  Leu Asp Asn Pro Asp Asn Glu Ser Ile Leu Glu Asp Ile Val Gln Thr      610                 615                 620  Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Lys Lys Arg Leu Glu Asn  625                 630                 635                 640  Tyr Lys Asp Leu Phe Thr Glu Ser Gln Leu Lys Lys Leu Tyr Arg Arg                  645                 650                 655  His Tyr Thr Gly Trp Gly Arg Leu Ser Ala Lys Leu Ile Asn Gly Ile              660                 665                 670  Arg Asp Lys Glu Ser Gln Lys Thr Ile Leu Asp Tyr Leu Ile Asp Asp          675                 680                 685  Gly Arg Ser Asn Arg Asn Phe Met Gln Leu Ile Asn Asp Asp Gly Leu      690                 695                 700  Ser Phe Lys Ser Ile Ile Ser Lys Ala Gln Ala Gly Ser His Ser Asp  705                 710                 715                 720  Asn Leu Lys Glu Val Val Gly Glu Leu Ala Gly Ser Pro Ala Ile Lys                  725                 730                 735  Lys Gly Ile Leu Gln Ser Leu Lys Ile Val Asp Glu Leu Val Lys Val              740                 745                 750  Met Gly Tyr Glu Pro Glu Gln Ile Val Val Glu Met Ala Arg Glu Asn          755                 760                 765  Gln Thr Thr Asn Gln Gly Arg Arg Asn Ser Arg Gln Arg Tyr Lys Leu      770                 775                 780  Leu Asp Asp Gly Val Lys Asn Leu Ala Ser Asp Leu Asn Gly Asn Ile  785                 790                 795                 800  Leu Lys Glu Tyr Pro Thr Asp Asn Gln Ala Leu Gln Asn Glu Arg Leu                  805                 810                 815  Phe Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Thr Gly Glu Ala              820                 825                 830  Leu Asp Ile Asp Asn Leu Ser Gln Tyr Asp Ile Asp His Ile Ile Pro          835                 840                845  Gln Ala Phe Ile Lys Asp Asp Ser Ile Asp Asn Arg Val Leu Val Ser      850                 855                 860  Ser Ala Lys Asn Arg Gly Lys Ser Asp Asp Val Pro Ser Leu Glu Ile  865                 870                 875                 880  Val Lys Asp Cys Lys Val Phe Trp Lys Lys Leu Leu Asp Ala Lys Leu                  885                 890                 895  Met Ser Gln Arg Lys Tyr Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly              900                 905                 910  Leu Thr Ser Asp Asp Lys Ala Arg Phe Ile Gln Arg Gln Leu Val Glu          915                 920                 925  Thr Arg Gln Ile Thr Lys His Val Ala Arg Ile Leu Asp Glu Arg Phe      930                 935                 940  Asn Asn Glu Leu Asp Ser Lys Gly Arg Arg Ile Arg Lys Val Lys Ile  945                 950                 955                 960  Val Thr Leu Lys Ser Asn Leu Val Ser Asn Phe Arg Lys Glu Phe Gly                  965                 970                 975  Phe Tyr Lys Ile Arg Glu Val Asn Asn Tyr His His Ala His Asp Ala              980                 985                 990  Tyr Leu Asn Ala Val Val Ala Lys Ala Ile Leu Thr Lys Tyr Pro Gln          995                    1000             1005  Leu Glu Pro Glu Phe Val Tyr Gly Asp Tyr Pro Lys Tyr Asn Ser Tyr      1010                    1015            1020  Lys Thr Arg Lys Ser Ala Thr Glu Lys Leu Phe Phe Tyr Ser Asn Ile  1025                    1030            1035                1040  Met Asn Phe Phe Lys Thr Lys Val Thr Leu Ala Asp Gly Thr Val Val                      1045            1050                1055  Val Lys Asp Asp Ile Glu Val Asn Asn Asp Thr Gly Glu Ile Val Trp                  1060           1065                 1070  Asp Lys Lys Lys His Phe Ala Thr Val Arg Lys Val Leu Ser Tyr Pro              1075            1080                1085  Gln Asn Asn Ile Val Lys Lys Thr Glu Ile Gln Thr Gly Gly Phe Ser          1090            1095                1100  Lys Glu Ser Ile Leu Ala His Gly Asn Ser Asp Lys Leu Ile Pro Arg  1105                1110                1115                1120  Lys Thr Lys Asp Ile Tyr Leu Asp Pro Lys Lys Tyr Gly Gly Phe Asp                  1125                1130                1135  Ser Pro Ile Val Ala Tyr Ser Val Leu Val Val Ala Asp Ile Lys Lys              1140                1145                1150  Gly Lys Ala Gln Lys Leu Lys Thr Val Thr Glu Leu Leu Gly Ile Thr          1155                1160                1165  Ile Met Glu Arg Ser Arg Phe Glu Lys Asn Pro Ser Ala Phe Leu Glu      1170                1175                1180  Ser Lys Gly Tyr Leu Asn Ile Arg Ala Asp Lys Leu Ile Ile Leu Pro  1185                1190                1195                1200  Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Arg Arg Leu Leu Ala                  1205                1210                1215  Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Thr Gln              1220                1225                1230  Phe Met Lys Phe Leu Tyr Leu Ala Ser Arg Tyr Asn Glu Ser Lys Gly          1235                1240                1245  Lys Pro Glu Glu Ile Glu Lys Lys Gln Glu Phe Val Asn Gln His Val      1250                1255                1260  Ser Tyr Phe Asp Asp Ile Leu Gln Leu Ile Asn Asp Phe Ser Lys Arg  1265                1270                1275                1280  Val Ile Leu Ala Asp Ala Asn Leu Glu Lys Ile Asn Lys Leu Tyr Gln                  1285                1290                1295  Asp Asn Lys Glu Asn Ile Ser Val Asp Glu Leu Ala Asn Asn Ile Ile              1300                1305                1310  Asn Leu Phe Thr Phe Thr Ser Leu Gly Ala Pro Ala Ala Phe Lys Phe          1315               1320                 1325  Phe Asp Lys Ile Val Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val      1330                1335                1340  Leu Asn Ser Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr  1345                1350                1355                1360  Arg Ile Asp Leu Gly Lys Leu Gly Glu Asp                  1365                1370  SEQ ID NO: 4211  atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg   60  atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc  120  cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa  180  gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt  240  tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga  300  cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga  360  aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa  420  aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat  480  atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat  540  gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct  600  attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga  660  cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat  720  ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa  780  gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg  840  caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt  900  ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca  960  atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga 1020  caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca 1080  ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta 1140  gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc 1200  aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat 1260  gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt 1320  gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt 1380  cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa 1440  gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa 1500  aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt 1560  tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt 1620  tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc 1680  gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt 1740  tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt 1800  attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt 1860  ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct 1920  cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga 1980  cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta 2040  gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat 2100  agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta 2160  catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact 2220  gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt 2280  attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt 2340  atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct 2400  gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga 2460  gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac 2520  attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct 2580  gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa 2640  aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta 2700  acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa 2760  ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat 2820  actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct 2880  aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat 2940  taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa 3000  tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa 3060  atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct 3120  aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc 3180  cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt 3240  gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta 3300  cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt 3360  gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct 3420  tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt 3480  aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac 3540  tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa 3600  tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta 3660  caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt 3720  cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag 3780  cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt 3840  attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa 3900  ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct 3960  cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa 4020  gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt 4080  gatttgagtc agctaggagg tgac 4104  SEQ ID NO: 4212  Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val  1               5                   10                  15  Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe              20                  25                  30  Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile          35                  40                  45  Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu      50                  55                  60  Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys  65                  70                  75                  80  Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser                  85                  90                  95  Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys              100                 105                 110  His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr          115                 120                 125  His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp      130                 135                 140  Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His  145                 150                 155                 160  Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro                  165                 170                 175  Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr              180                 185                 190  Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala          195                 200                 205  Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn      210                 215                 220  Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn  225                 230                 235                 240  Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe                  245                 250                 255  Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp              260                 265                 270  Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp          275                 280                 285  Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp      290                 295                 300  Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser  305                 310                 315                 320  Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys                  325                 330                 335  Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe              340                 345                 350  Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser          355                 360                 365  Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp      370                 375                 380  Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg  385                 390                 395                 400  Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu                  405                 410                 415  Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe              420                 425                 430  Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile          435                 440                 445  Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp      450                 455                 460  Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu  465                 470                 475                 480  Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr                  485                 490                 495  Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser              500                 505                 510  Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys          515                 520                 525  Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln      530                 535                 540  Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr  545                 550                 555                 560  Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp                  565                 570                 575  Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly              580                  585                590  Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp          595                 600                 605  Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr      610                 615                 620  Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala  625                 630                 635                 640  His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr                  645                 650                 655  Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp              660                 665                 670  Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe          675                 680                 685  Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe       690                695                 700  Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu  705                 710                 715                 720  His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly                  725                 730                 735  Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly              740                 745                 750  Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln          755                 760                 765  Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile      770                 775                 780  Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro  785                 790                 795                 800  Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu                  805                 810                 815  Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg              820                  825                830  Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys          835                 840                 845  Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg      850                 855                 860  Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys  865                 870                 875                 880  Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys                  885                 890                 895  Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp              900                 905                 910  Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr          915                 920                 925  Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp      930                 935                 940  Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser  945                 950                 955                 960  Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg                  965                 970                 975  Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val              980                 985                 990  Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe          995                 1000                1005  Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys      1010                1015                1020  Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser  1025                1030                1035                1040  Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu                  1045                1050                1055  Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile              1060                1065                1070  Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser          1075                1080                1085  Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly      1090                1095                1100  Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile  1105                1110                1115                1120  Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser                  1125                1130                1135  Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly              1140                1145                1150  Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile          1155                1160                1165  Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala      1170                1175                1180  Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys  1185                1190                1195                1200  Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser                  1205                1210                1215  Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr              1220                1225                1230  Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser          1235                1240                1245  Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His      1250                1255                1260  Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val  1265                1270                1275                1280  Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys                  1285                1290                1295  His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu              1300                1305                1310  Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp          1315                1320                1325  Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp      1330                1335                1340  Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile  1345                1350                1355                1360  Asp Leu Ser Gln Leu Gly Gly Asp                  365  

The invention claimed is:
 1. A fusion polypeptide consisting of (i) a polypeptide which has at least 95% sequence identity to SEQ ID NO: 4212 and (ii) a heterologous sequence.
 2. The fusion polypeptide of claim 1, wherein the polypeptide of (i) consists a sequence which has at least 99% sequence identity to SEQ ID NO:
 4212. 3. The fusion polypeptide of claim 1, wherein the polypeptide of (i) consists of the sequence of SEQ ID NO:
 4212. 4. The fusion polypeptide of claim 1, wherein the heterologous sequence of the fusion polypeptide comprises a histidine tag, a glutathione S-transferase (GST) tag, a signal sequence, or a leader sequence.
 5. A composition comprising (a) a fusion polypeptide of claim 1, and (b) a pharmaceutically acceptable vehicle. 